Overexpression of the tomato SlLEA_2-26 gene enhances the tolerance to drought and salt stresses in Arabidopsis thaliana

Overexpression of the tomato SlLEA_2-26 gene enhances the tolerance to drought and salt stresses in Arabidopsis thaliana | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Overexpression of the tomato SlLEA_2-26 gene enhances the tolerance to drought and salt stresses in Arabidopsis thaliana Zhehua Yan, Yu Lei, Xuan Zou, Sijie Wang, Yanxin Yang, Dongjing Yang, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8801747/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Late embryogenesis abundant (LEA) proteins are pivotal in conferring cellular tolerance to abiotic stresses and sustaining plant growth and development. However, systematic functional characterization of the tomato SlLEA_2 gene family remains limited. To elucidate the role of tomato SlLEA_2–26 in abiotic stress responses, this study cloned its full-length cDNA. Quantitative real-time PCR (qRT-PCR) analysis revealed that SlLEA_2–26 exhibits predominant expression in flowers and fruits, and is strongly induced by drought, salt, Cu 2+ , and Pb 2+ stresses. Three homozygous Arabidopsis T 3 SlLEA_2-26 -overexpression lines were generated and confirmed via genomic PCR. Under drought and salt stress, T 3 Arabidopsis SlLEA_2-26 -overexpressing lines demonstrated significantly enhanced seed germination rates, root elongation, and fresh weights wild type (WT) plants, indicating improved stress tolerance during early seedling development. Furthermore, transgenic plants accumulated higher levels of soluble sugar and proline, and displayed elevated antioxidant enzyme activity compared to the WT, whereas contents of malondialdehyde (MDA) and reactive oxygen species (ROS) were markedly reduced relative to WT. qRT-PCR analysis confirmed the significant upregulation of SlLEA_2–26 in transgenic lines under drought and salt stress conditions, accompanied by elevated expression of AtP5CS1 , AtCSD1 , AtRD29A , AtRD26 , and AtNCED3 . Collectively, these results demonstrate that SlLEA_2–26 overexpression enhances drought and salt stress tolerance in Arabidopsis by promoting the accumulation of osmoregulatory substances, augmenting antioxidant defense capacity, and activating stress-responsive genes expression. This study provides a theoretical foundation and valuable genetic resources for breeding stress-tolerant tomatoes and other crops. Abiotic stress Late embryogenesis abundant protein SlLEA_2–26 Stress tolerance Tomato Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Headings SlLEA_2–26 gene was cloned from the tomato cultivar 1436. The multiple environmental stresses significantly increased the expression of SlLEA_2–26 gene in tomato leaves. Overexpression of SlLEA_2–26 significantly improved the salt and drought stress tolerance in transgenic Arabidopsis lines. 1. Introduction Tomato ( Solanum lycopersicum L.) is a globally cultivated crop of high nutritional value (Ali MY et al. 2020). It serves as the richest dietary source of lycopene, a carotenoid with an antioxidant capacity 100 times that of vitamin E (Burton-Freeman B and Sesso HD 2014). Lycopene is associated with delayed cellular aging and a reduced risk of certain cancers and cardiovascular diseases (Petyaev IM 2016; Perveen R et al. 2015). Furthermore, tomatine present in tomatoes exhibits antibacterial and anti-inflammatory properties. However, drought and salinization constitute major environmental constraints that impede crop growth, significantly reduce crop yields, and limit agricultural and economic development. Late embryogenesis abundant (LEA) proteins, widely distributed in plants, accumulate abundantly during the late stages of embryo development (Banerjee A and Roychoudhury A 2016; Dure L 3rd et al. 1981). These proteins can chelate metal ions (Grelet J et al. 2005), protect enzyme activity (Reyes JL et al. 2008), and mitigate cellular damage under drought stress (Tolleter D et al. 2007). Initially isolated from Gossypium hirsutum cotyledons during embryogenesis, LEA proteins have since been studied in Arabidopsis thaliana (Hundertmark M and Hincha DK 2008), Oryza sativa (Rodríguez-Valentín R et al. 2014), Cucumis sativus (Celik Altunoglu Y et al. 2016), and Zea mays (Liu Y et al. 2013). By shielding plant cells from dehydration-induced damage caused by drought and salinity, LEA proteins play a critical role in sustaining normal plant growth and development (Zaman Khan N et al. 2020). As the LEA protein family continues to expand, its classification framework has been progressively refined. Currently, LEA proteins are primarily categorized into eight subgroups, including dehydrins (DHN), seed maturation proteins (SMP), and LEA_1 through LEA_6. Most of these proteins are characterized by high hydrophilicity. A notable exception is the LEA_2 subgroup, which is enriched in hydrophobic residues, rendering it the least hydrophilic member of the family. According to an earlier classification proposed by Battaglia’s group, LEA proteins were divided into seven groups (groups 1–7). Among these, groups 1, 2, 3, 4, 6, and 7 are considered typical LEA proteins due to their marked hydrophilicity and conserved motifs. In contrast, group 5 is classified as atypical because of its low sequence homology and significantly higher proportion of hydrophobic amino acids. Notably, LEA_2 was originally assigned to group 5 in this scheme (Battaglia M et al. 2008), its established crucial role in plant abiotic stress responses (Galau GA et al. 1993). Research indicates that nearly all LEA gene promoters contain cis-regulatory elements such as ABRE, MBS, W-box, and TAC, which are integral to the plant stress-response network (Magwanga RO et al. 2018). Moreover, miRNA-mediated reprogramming of gene expression serves as a key defense mechanism under drought stress (Ferdous J et al. 2015). Functioning primarily at the post-transcriptional level, numerous miRNAs regulate plant adaptation to abiotic stresses (Grivna ST et al. 2006). For instance, in cotton, 48 distinct miRNAs are predicted to target 63 LEA_2 genes, accounting for approximately 40% of this subfamily, with several genes being targeted by more than six miRNAs. These miRNAs also regulate multiple known drought-responsive genes and target stress-related cis-elements, including ABRE, DRE/CRT, MYBS, and LTRE (Magwanga RO et al. 2018). Through this coordinated regulation of stress-associated genes and regulatory motifs, miRNAs play a vital role in plant responses to drought and salinity. This positions LEA_2 as a pivotal integrator of stress signals within the regulatory network, acting as both a key effector and a synergistic regulator in plant abiotic stress defense. LEA_2 proteins function as crucial molecular components in plant responses to abiotic stress, and their roles have been extensively characterized. Genes encoding LEA_2 proteins, identified across diverse species, are consistently involved in regulating plant adaptation to drought and high salinity. For example, the expression of CaLEA6 in pepper and AdLEA in peanut is significantly upregulated in response to NaCl and PEG treatments, indicating their active participation in coping with salt-induced and drought-induced water deficit (Kim HS et al. 2005; Sharma A et al. 2016). Furthermore, heterologous expression of SmLEA2 from Salvia miltiorrhiza enhances Escherichia coli growth under salt and drought stress. Similarly, transgenic plants overexpressing this gene show increased stress tolerance, whereas its suppression leads to greater susceptibility (Wang HQ et al. 2017). Notably, maize Rab28 is classified within group 5 alongside LEA_2 proteins. Overexpression of the Rab28 gene significantly improves osmotic stress tolerance in maize (Amara I et al. 2013). Mechanistically, LEA_2 proteins enhance stress resistance by modulating stress-related signaling pathways. For instance, in transgenic Arabidopsis overexpressing the cotton gene CotAD_24498 , drought stress significantly upregulates the expression of ABF4 and RD29A (Magwanga RO et al. 2018). The transcription factor ABF4, a central component of the ABA signaling pathway, activates a suite of drought-responsive genes by binding to ABRE cis-elements within their promoters. Concurrently, the elevated expression of RD29A , a well-established drought stress marker, directly reflects a heightened cellular capacity to respond to water deficit. Thus, CotAD_24498 likely strengthens drought tolerance by constitutively activating the ABA signaling network and its downstream targets. In addition, under drought stress, tobacco plants overexpressing the peanut AdLEA gene significantly upregulate the expression of the NtERD10c gene. ERD10c encodes a hydrophilic group 2 LEA protein, which contributes to dehydration tolerance by binding water molecules, stabilizing labile enzymes, and protecting macromolecular structures (Kovacs D et al. 2008). Transgenic plants may synthesize increased amounts of protective molecular chaperones, thereby more effectively mitigating water loss under dehydration or drought stress (Chakrabortee S et al. 2007). Although the overexpression of LEA_2 in plants can enhance stress tolerance, studies on the function of tomato LEA_2 genes in response to abiotic stress are still relatively limited. In this study, a tomato SlLEA_2-26 gene was cloned from cultivar 1436, and transgenic Arabidopsis overexpression lines were constructed. Additionally, its expression profiles were analyzed across different tomato tissues and under various stress conditions. The tolerance of transgenic Arabidopsis to salt and drought stresses was assessed, and the expression levels of stress-related genes were measured. These results elucidated the molecular mechanism by which SlLEA_2-26 responds to abiotic stress, provided a theoretical basis and genetic resources for stress-resistant breeding in tomatoes and other crops. 2. Materials and methods 2.1. Plant materials and growing conditions Arabidopsis thaliana ecotype Colombia-0 (Col-0) and tomato cultivar 1436 were employed as wild types (WT). Various tomato tissues, including seeds, cotyledons, hypocotyls, roots, stems, leaves, flowers, and fruits, were collected from plants grown under normal conditions for tissue-specific expression profiling. To assess mRNA expression levels of the SlLEA_2-26 gene under different abiotic stresses, 3-week-old tomato plants were grown in 1/2 Hoagland nutrient solution supplemented with 200 mM NaCl, 15% PEG6000, 800 μM CdSO 4 , 800 μM CuSO 4 , 5 mM Pb(CH 3 COO) 2 , 75 μM ABA, respectively, or subjected to 44 ℃ and 4 ℃ treatments. At 0, 1, 3, 6, 12, 24, and 48 h, the third and fourth leaves from the bottom were collected as experimental material for further quantitative real-time PCR analysis. 2.2. Phylogenetic and sequence alignment analysis of SlLEA_2-26 Homologous protein sequences were retrieved using the NCBI-BLAST tool. Multiple sequence alignment of the protein sequences was performed with DNAMAN 5.2.2.0 software. A phylogenetic tree was constructed with MEGA 7.1 software based on the Neighbor-Joining (NJ) method. The subcellular localization of the SlLEA_2-26 protein was predicted using WoLF PSORT (https://wolfpsort.hgc.jp/). 2.3. Quantitative real-time PCR (qRT-PCR) analysis of gene expression Total RNA was extracted using TriQuick reagent (Solarbio, China), and cDNA was synthesized with a reverse transcription kit (Biosharp, Anhui, China) for subsequent qRT-PCR analysis. The specific primers designed via Primer-BLAST on the NCBI website are provided in Table S1. The reagent used in the qRT-PCR reaction was 2×M5 HiPer SYBR Premix EsTaq (with Tli RNaseH). SlActin was used as the internal control gene for qRT-PCR analysis. The relative expression levels of SlLEA_2-26 were calculated using the 2 - △△ CT method. 2.4. Generation of SlLEA_2-26- overexpression lines in Arabidopsis The full-length SlLEA_2-26 gene was inserted into the pCAMBIA1302 driven by a stress-inducible promoter SWPA2 to generate the recombinant vector pCAMBIA1302- SlLEA_2-26 . The construct was transformed into Agrobacterium tumefaciens GV3101 for Col-0 transformation via floral dipping (Clough SJ and Bent AF 1998). T 3 homozygous overexpression lines were selected via hygromycin resistance (50 mg·L -1 ) and verified through genomic PCR. Three resulting T 3 transgenic lines (OE6, OE9, OE11) were randomly selected for subsequent functional analysis. 2.5. Abiotic stress tolerance assay in T 3 transgenic Arabidopsis For germination assays, seeds of both WT and OEs were cultured on 1/2 MS solid medium under osmotic and salt stress conditions, imposed by supplementation with 200 mM mannitol or 100 mM NaCl, respectively. After 7 days of incubation, the germination rates were calculated (germination was defined as the appearance of the radicle). For root growth tests, 5-day-old seedlings with a similar root length were transferred to 1/2 MS solid medium supplemented with 225 mM mannitol and 125 mM NaCl, respectively. The root lengths of the vertically cultured seedlings were measured after 7 days of growth. To assess the potential of SlLEA_2-26 overexpression in improving salt and drought tolerance at the whole plant level, 20-day-old uniform seedlings of WT and OEs plants were withheld from irrigation 14 days and then rewatered for 3 day, or subjected to irrigation with 225 mM NaCl solution for 15 days. Leaves were collected at 0, 7, 14 days after irrigation cessation, and 3 days after resumption. Under salt stress, leaves were sampled at 0, 5, 10, 15 days for further analysis. 2.6. Determination of MDA, H 2 O 2 , O 2 ·- , soluble sugar and proline content The Thio-barbituric acid (TBA) method was used to measure malondialdehyde (MDA) content (Wang WB et al. 2009). Evaluation of plant hydrogen peroxide (H 2 O 2 ) content was performed by the xylenol orange method (Bindschedler LV et al. 2001). The superoxide anion (O 2 ·- ) content was determined according to the hydroxylamine oxidation method (Kaur N et al. 2016). Soluble sugar and proline contents were determined using corresponding assay Kits (Sangon Biotech, Shanghai, China). 2.7.Measurement of antioxidant enzyme activity The activities of superoxide dismutase (SOD) and peroxidase (POD) were determined using the nitroblue tetrazolium (NBT) photochemical reduction and guaiacol methods, respectively (Chen TZ and Zhang BL 2016). SOD activity was quantified as the amount of enzyme causing 50% inhibition of NBT reduction. Catalase (CAT) and ascorbate peroxidase (APX) activities were assayed using spectrophotometric methods as previously reported (Wang WB et al. 2009). The units of all antioxidant enzymes activity were represented as U·mg -1 protein. 2.8. Determination of relevant gene expression levels RNA was extracted from stress-treated samples using TriQuick reagent (Solarbio, China), and cDNA was synthesized using a reverse transcription kit (Biosharp, Anhui, China). The expression levels of SlLEA_2-26 , AtP5CS1 , AtCSD1 , AtNCED3 , AtRD26 , and AtRD29A were determined by qRT-PCR using AtActin genes as internal reference gene. The qRT-PCR primers were listed in Supplementary Table S1. 2.9. Statistical analysis All statistical analyses in this study were performed using IBM SPSS 25.0 (IBM Corp, USA). Data represent mean ± SE of three biological replicates, and statistical differences were assessed using analysis of variance (ANOVA) and independent samples t -test. The significant differences at P < 0.05 and extremely significant differences at P < 0.01 between WT and OE Arabidopsis lines were determined ( *P < 0.05 and **P < 0.01). 3. Results 3.1. Cloning and sequence analysis of SlLEA_2-26 gene In our previous study, the tomato LEA_2 gene family was systematically analyzed. Based on its chromosomal location, the gene investigated in this work was identified and named (data not shown). The SlLEA_2-26 cDNA was isolated from tomato cultivar 1436 (GenBank Accession No. PP130721). This gene has a full-length of 666 bp encoding 221 amino acids in which contains abundant 27 leucine residues, while tryptophan is present only once. The SlLEA_2-26 protein possesses a transmembrane structure and is predicted as an unstable, basic, and hydrophobic protein. WoLF PSORT analysis predicted a cytoplasmic localization for SlLEA_2-26. Tomato SlLEA_2-26 was the most closely related to the LEA homologs of Quillaja saponaria through phylogenetic analysis (Fig. 1a). Multiple sequence alignment indicated that SlLEA_2-26 protein contains the typical LEA_2 domain (Fig. 1b). Taken together, these results suggested that SlLEA_2-26 is a functional LEA_2 protein. 3.2. Tissue-specific and stress-induced expression profiles analysis of SlLEA_2-26 in tomato qRT-PCR analysis measured SlLEA_2-26 expression in seeds, cotyledons, hypocotyls, roots, stems, leaves, flowers, and fruits. The results indicated that the SlLEA_2-26 gene is differentially expressed across various tissues, with significantly higher expression levels predominantly observed in flowers and fruits. In contrast, the relative expression of SlLEA_2-26 was found to be minimal in seeds at post-germination 4 days (Fig. 2a). To elucidate the response pattern of SlLEA_2-26 to various stresses, its expression dynamics under treatments of NaCl, PEG, ABA, extreme temperatures, and four metal ions were further analyzed by qRT-PCR (Fig. 2b). Under eight different abiotic stresses, the expression levels of SlLEA_2-26 generally showed an initial increase followed by a decrease over time. The gene exhibited strong responses to PEG, NaCl, copper, and lead stresses, and lower responses to extreme temperatures, cadmium, and ABA stresses. The SlLEA_2-26 gene could be upregulated and reached a peak at 24 h under drought stress, while these peaks occurred at 6 h and 3 h under copper and lead stress, respectively. However, SlLEA_2-26 expression exhibited a rapid but short-lived response to lead stress, primarily functioning during the early stages of stress exposure. Additionally, under NaCl stress, the relative expression level of the SlLEA_2-26 sharply increased to a peak at 1 h, followed by an overall downward trend while remaining significantly higher than that before the treatment. This result indicated that the SlLEA_2-26 exhibits a rapid and sustained response to salt stress. In summary, the induced expression of the SlLEA_2-26 gene exhibited a tissue specificity. The different expression profile under the various stress suggested that the SlLEA_2-26 gene can rapid or sustained response to adverse environment. 3.3. Overexpression of the SlLEA_2-26 enhances seed germination rate and root elongation under drought and salt stress Three homozygous SlLEA_2-26 -overexpressing Arabidopsis lines (OE6, OE9, and OE11) verified by genomic PCR were selected to investigate whether overexpression of SlLEA_2-26 can affect seed germination. Under normal growth conditions, seed germination rates of WT and SlLEA_2-26 overexpressing lines were consistent with their phenotypes and showed no significant differences (Fig. 3a,b). However, the OEs exhibited significantly higher germination rates than that of WT under mannitol and NaCl stresses. For instance, the germination rate under NaCl stress was approximately 88% for OEs seeds, in contrast to 72% for WT. The root system of plants is a key organ to acquire nutrients from the external environment, and the stress tolerance of Arabidopsis at the seedling stage can be evaluated by root elongation under adverse environment (Jisha KC et al. 2013). No significant difference in growth phenotype (Fig. 3c), fresh weight (Fig. 3d), or root length (Fig. 3e) was detected between WT and OEs seedlings under normal growth conditions. In contrast, under osmotic stress induced by mannitol and ionic stress imposed by NaCl, OEs seedlings demonstrated significantly enhanced root elongation and increased fresh weight, respectively, relative to WT. In conclusion, heterologous overexpression of SlLEA_2-26 gene significantly promoted seed germination and root elongation in Arabidopsis under drought and salt stress, and contributed to maintaining higher fresh weight in OE plants. 3.4. Overexpression of SlLEA_2-26 enhances drought stress tolerance in adult Arabidopsis plants The effects of SlLEA_2-26 overexpression on drought tolerance in adult Arabidopsis plants were further investigated through potted-plant water control experiment. Under normal growth conditions WT and Arabidopsis plants exhibited comparable growth phenotypes. During drought stress, however, WT plants exhibited leaves purpling, and overall darkening, while a portion of leaves in OEs remained green. Three days after rewatering, WT plants showed more severe wilting and reduction in plant size compared to the OE lines (Fig. 4a). Proline and soluble sugars, as typical osmoregulatory substances, are important indicators for evaluating stress resistance. Their accumulation under stress conditions helps maintain cellular osmotic pressure and alleviates osmotic damage (Waadt R et al. 2022). Before treatment, soluble sugar and proline contents showed no significant differences between WT and OE Arabidopsis plants (Fig. 4b,c). As drought treatment duration extension, soluble sugar and proline contents in the OEs gradually increased and remained significantly higher than those in WT. At drought 14 th day, soluble sugar content in lines OE6, OE9, and OE11 reached levels as high as 37.28, 37.05, and 34.11 mg·g -1 FW, respectively (Fig. 4b). After rewatering 3 days, the contents of these osmoregulatory substances decreased, while those in OEs lines still exhibited significantly higher levels than WT (Fig. 4b,c). The results indicated that SlLEA_2-26 -overexpression in Arabidopsis improves the drought stress tolerance by promoting the accumulation of osmotic regulatory substances. Drought and salinity stress disrupted cellular redox balance, which led to the accumulation of reactive oxygen species (ROS) and thereby induced oxidative damage in plants (Li SF et al. 2025). During drought and rehydration periods, the MDA, H 2 O 2 and O 2 ·- levels in transgenic Arabidopsis were significantly lower than those in WT plants (Fig. 4h-j). The difference became more significant after 14 days of drought, with MDA content in OE Arabidopsis plants reduced by approximately 36% relative to WT. (Fig. 4h). Additionally, all the SOD, POD, APX, and CAT enzyme activities in the OE lines gradually increased and were significantly higher than those in WT under drought stress (Fig. 4d-g). Specifically, SOD activity in lines OE6, OE9, and OE11 was 1.52-, 1.43-, and 1.5-fold higher than in WT at drought 14 th day, respectively (Fig. 4d). These findings suggested that overexpression of SlLEA_2-26 gene in OEs improves the antioxidant enzymes activity, reduces ROS accumulation and membrane lipid peroxidation damage, and ultimately enhances the stress tolerance to drought. 3.5. Drought stress upregulates the expression of stress-responsive genes in transgenic Arabidopsis To investigate the molecular mechanism underlying the drought tolerance of SlLEA_2-26 , the relative expression levels of SlLEA_2-26 and the stress-responding genes AtP5CS1 , AtCSD1 , AtNCED3 , AtRD26 , and AtRD29A were detected in WT and OE plants under drought stress. As expected, the mRAN accumulation of SlLEA_2-26 in OEs continuously increased under drought condition (Fig. 5a). The expression patterns of above five stress-responsive genes in OEs resembled that of SlLEA_2-26 , peaking at 14 days under drought stress with the extremely significant difference compared to the WT plants (Fig. 5b-f). While AtP5CS1 expression in transgenic plants reached approximately twice the WT level at drought 14 th day, the expression of AtCSD1 in lines OE6, OE9, and OE11 was 1.04, 1.7, and 0.68 times higher than WT, respectively (Fig. 5b,c). Their expression levels slightly decreased after 3 days of rewatering. These results indicated that heterologous expression of SlLEA_2-26 can induce the upregulation of stress-associated genes in Arabidopsis under drought stress. 3.6. Overexpression of SlLEA_2-26 enhances salt stress tolerance in transgenic Arabidopsis To further investigate the effect of SlLEA_2-26 overexpression on stress tolerance to salt, WT and OE Arabidopsis plants were subjected to irrigation with 225 mM NaCl for 15 days.No significant differences were observed in growth phenotype, soluble sugar and proline content, ROS content, and antioxidant enzyme activities between OE and WT plants under normal growth condition (Fig. 6a-j). However, the WT plants showed more severe wilting than OEs under salt stress, the majority of leaves in the OEs remained green. (Fig. 6a). Throughout the entire salt stress process, the contents of soluble sugar and proline continuously increased, and both remained significantly higher level in OEs than that in the WT plants (Fig. 6b,c). At day 5 of stress, the soluble sugar content in the overexpression lines increased markedly to 17 mg·g -1 FW (Fig. 6b). As NaCl stress treatment time extension, the contents of MDA, H 2 O 2 , and O 2 ·- gradually increased, while those in the OE lines consistently exhibited significantly lower levels than that in the WT (Fig. 6h-j). At 15 th day of salt stress, the H 2 O 2 content in the OEs was approximately 80% that of the WT (Fig. 6i). Meanwhile, the O 2 ·- content in lines OE6, OE9, and OE11 was reduced by 23.03%, 25%, and 31.65%, respectively, compared to the WT (Fig. 6j). Similarly, the activities of SOD, POD, APX, and CAT rapidly increased after salt stress, and higher activity levels of antioxidant enzymes were presented in OEs plants than those in the WT (Fig. 6d-g). Remarkably, the OE lines exhibited higher POD and CAT activities, with their activities rising sharply compared to the WT after stress exposure. Moreover, this difference was most significant at 15 days after salt stress, with POD activity elevated by over 50% and CAT activity increased nearly two folds, respectively (Fig. 6e,g). The results above indicated that SlLEA_2-26 -overexpression in Arabidopsis enhances salt tolerance by promoting the accumulation of osmotic regulatory substances, increasing the antioxidant enzyme activities, and alleviating oxidative damage caused by NaCl stress. 3.7. Salt stress upregulates the expression of stress-responsive genes in transgenic Arabidopsis The relative expression levels of SlLEA_2-26 and stress-responding genes were further detected in OE lines and WT plants under NaCl treatment to reveal the molecular mechanism of salt stress tolerance. As expected, salt stress significantly induced the upregulation of SlLEA_2-26 gene in OE Arabidopsis plants with the extension of stress treatment (Fig. 7a). In addition, the mRNA levels of five stress- associated genes including AtP5CS1 , AtCSD1 , AtNCED3 , AtRD26 , and AtRD29A in Arabidopsis continuously increased with the extension of stress duration (Fig. 7b-f). Meanwhile, the OE plants exhibited significant higher expression level of stress-responding genes than that in WT. After 15 days of salt stress, transgenic plants showed a 59-89% increase in AtP5CS1 expression relative to WT, while AtCSD1 expression reached about 2.5 times the WT level (Fig. 7b,c). In conclusion, overexpression of SlLEA_2-26 in Arabidopsis can induce the upregulation of stress-responsive genes under salt stress. 4. Discussion In this study, SlLEA_2-26 gene exhibited distinct tissue-specific expression patterns, with significantly higher transcript levels detected in flowers and fruits compared to other tissues (Fig. 2a). These observations align with prior reports indicating that Ipomoea pescaprae IpLEA (Zheng JX et al. 2019) and rice OsEm1 (Yu J et al. 2016) display elevated transcriptional level in flowers and developing seeds, consistent with the conserved functional characteristics of LEA genes. SlLEA_2-26 gene demonstrated differential responsiveness to diverse abiotic stresses, exhibiting marked upregulation under drought and salt stress conditions. Under heat and cold stress, transcript level reached up to six-fold higher than those under normal growth conditions, whereas its responsiveness to ABA was comparatively weak (Fig. 2b). The observed stress response patterns of SlLEA_2-26 gene to abiotic stresses are consistent with previous studies (Park SC et al. 2011; Zheng JX et al. 2019). Collectively, these results indicated that SlLEA_2-26 play a positive role under multiple stress conditions, thereby prompting further functional validation in transgenic Arabidopsis lines. Drought and salt stress can significantly impair seed germination efficiency and inhibit seedling root elongation, both of which serve as critical physiological indicators for evaluating plant stress tolerance (Henry A 2013). Consistent with these findings, heterologous expression of cotton GhLEA_2 in Arabidopsis and overexpression of Salvia miltiorrhiza SmLEA2 were demonstrated to significantly promote root growth in transgenic plants (Magwanga RO et al. 2018; Wang HQ et al. 2017). Similarly, overexpression of rice OsEm1 has been shown to enhance drought and salt tolerance in rice seedlings (Yu J et al. 2016). In this study, under drought and salt stress conditions, transgenic Arabidopsis lines overexpressing SlLEA_2-26 exhibited significantly higher seed germination rates, increased root lengths, and greater fresh weights compared to the WT plants (Fig. 3a-e). Furthermore, phenotypic analysis revealed pronounced morphological differences between WT and OE plants, with the latter displaying enhanced tolerance during stress stages (Fig. 4a, Fig. 6a). These observations collectively indicate that SlLEA_2-26 overexpression confers improved drought and salt tolerance in Arabidopsis during both germination and seedling establishment phases. Drought and salinity can induce osmotic stress in plants, prompting cellular accumulation of osmoregulatory substances including soluble sugars and proline to mitigate adverse effects (Bao YQ et al. 2025). Proline serves a multifaceted role in stress adaptation: it can not only maintain the osmotic balance between cells and the environment but also act as a signaling molecule to activate various responses related to abiotic stress (Kavi Kishor PB and Sreenivasulu N 2014; Natarajan SK et al. 2012). In this study, under both drought and salt stress, transgenic Arabidopsis lines overexpressing SlLEA_2-26 exhibited substantial accumulation of soluble sugars and proline, with levels significantly exceeding those in WT plants (Fig. 4b,c; Fig. 6b,c). These findings indicate that SlLEA_2-26 overexpression enhances the synthesis of osmoregulatory compounds under drought and salt condition, thereby improving stress tolerance through osmotic adjustment. Both the proteins encoded by foxtail millet SiLEA14 and tomato SlLEA_2-26 belong to atypical LEA proteins. Under drought or salt stress, proline and soluble sugar accumulation patterns in SiLEA14 - overexpressing foxtail mille t were consistent with those observed in this study, demonstrating conserved regulatory mechanisms. These proteins maintain cellular water balance by enhancing osmotic adjustment capacity and alleviating drought-induced cellular damage (Wang MZ et al. 2014), corroborating findings from studies on LEA genes overexpression in alfalfa (Jia HL et al. 2020; Ma WX et al. 2025), ginseng (Wang Q et al. 2024), and muskmelon (Aduse Poku S et al. 2020). Notably, proline expression levels reached exceptionally high values under salt stress, aligning with transcriptional analysis data (Fig. 7b). This observation further substantiates that SlLEA_2-26 exhibits a robust response to salt stress, highlighting its potential as a key regulator in salinity adaptation. Drought and salt stresses induce excessive accumulation of ROS in plants, which can trigger biomembrane system damage through lipid peroxidation, impairing photosynthesis, disrupting respiratory metabolism, and ultimately leading to plant mortality (Basu S et al. 2021). The levels of O 2 ·- and H 2 O 2 serve as direct indicators of ROS accumulation, whereas MDA content reflects the extent of membrane lipid peroxidation (Hasanuzzaman M and Fujita M 2022). During the long evolutionary process, Plants have evolved an antioxidant defense to scavenge ROS and mitigate oxidative damage, thereby maintaining physiological homeostasis. Under stress condition, antioxidant enzymes including SOD, APX, POD, and CAT are activated to counteract abiotic stresses (Sofo A et al. 2015). SOD catalyzes the dismutation of O 2 ·- into H 2 O 2 and O 2 , while APX, POD, and CAT collectively scavenge H 2 O 2 , thereby reducing ROS level and alleviating biomembrane damage. In this study, under both drought and salt stress condition, the contents of MDA, H 2 O 2 , and O 2 ·- in transgenic Arabidopsis lines were significantly reduced compared to WT plants, with the decline becoming more pronounced as the stress duration increased (Fig. 4h-j; Fig. 6h-j). This suggests diminished oxidative damage in transgenic plants. Concurrently, the activities of antioxidant enzymes including SOD, POD, APX, and CAT were significantly higher in OE Arabidopsis lines than those in WT plants (Fig. 4d-g, Fig. 6d-g). These findings align with previous reports, where heterologous overexpression of the muskmelon CmLEA-S gene in tobacco (Aduse Poku S et al. 2020) and the peanut AhLEA2 gene in Arabidopsis (Li C et al. 2022) similarly enhanced antioxidant enzyme activity and reduced MDA accumulation, accompanied by improved drought and salt stress tolerance in transgenic plants. Collectively, the data indicate that SlLEA_2-26 overexpression enhances antioxidant enzyme activity, thereby improving ROS scavenging capacity and effectively mitigating oxidative damage in transgenic Arabidopsis under drought and salt stress. Abiotic stress triggers the transcriptional activation of numerous stress-responsive genes. Zheng et al (2019) reported that heterologous overexpression of Ipomoea pescaprae IpLEA in Arabidopsis induced the expression of stress-associated genes including CSD1 , NCED3 , and RD29A , consequently enhancing abiotic stress tolerance in transgenic plants. To elucidate the molecular mechanisms underlying SlLEA_2-26 -mediated drought and salt stress responses, this study quantified the expression levels of five stress tolerance-related genes. The synthetase encoded by P5CS is the rate-limiting enzyme in the biosynthesis of proline (Amini S et al. 2015). CSD1 encodes SOD which catalyzes the dismutation of O 2 ·- into H 2 O 2 and O 2 (Cui LJ et al. 2015). NCED3 encodes a key enzyme in the biosynthesis of ABA, a pivotal phytohormone orchestrating abiotic stress responses such as drought (Nakashima K and Yamaguchi-Shinozaki K 2013). RD26 encodes an NAC transcription factor integral to ABA-mediated dehydration signaling pathways (Fujita M et al. 2004). RD29A responds to multiple abiotic stresses including drought, salinity, high temperature, and ABA, encoding a highly hydrophilic protein that protects cells from dehydration damage (Yamaguchi-Shinozaki K et al. 1992). In this study, all five stress-responsive genes exhibited significantly elevated expression levels in SlLEA_2-26 -overexpressing Arabidopsis compared to WT plants (Fig. 5b-f, Fig. 7b-f). Notably, the upregulation of AtP5CS1 and AtCSD1 correlated with increased proline accumulation and SOD activity under drought and salt stress (Fig. 4c-d, Fig. 5b-c, Fig. 6c-d, Fig. 7b-c). These findings indicate that overexpression of SlLEA_2-26 can induce the transcriptional activation of stress-responsive genes, thereby augmenting antioxidant enzyme activity and enhancing ROS scavenging efficiency. Concurrently, it activates ABA signaling pathway components, modulating Arabidopsis adaptive responses to environmental stress. Consistent with these observations, studies on tobacco overexpressing the peanut AdLEA demonstrated that upregulation of key stress response pathway genes confers enhanced abiotic stress tolerance in transgenic plants (Sharma A et al. 2016). This cross-species conservation underscores the pivotal role of LEA proteins in orchestrating plant stress adaptation. In conclusion, the overexpression of the SlLEA_2-26 gene in Arabidopsis triggers the upregulation stress-responsive genes under drought and salt stress, thereby mediating plant stress tolerance through dual regulatory pathways. Firstly, it promotes the biosynthesis of osmolytes such as proline via osmoprotectant synthesis, thereby maintaining cellular osmotic equilibrium and water homeostasis. Secondly, it synergistically enhances the activity of the antioxidant enzyme system, mitigating ROS-induced membrane damage and alleviating stress-related cellular dysfunction. Collectively, these mechanisms confer improved abiotic stress tolerance in transgenic plants. This study characterized the functional role of the SlLEA_2-26 gene by analyzing stress responses in transgenic Arabidopsis, and demonstrated its positive contribution to abiotic stress adaptation. Future research will focus on dissecting the regulatory networks of SlLEA_2-26 in tomato to elucidate its species-specific functions during abiotic stress. Abbreviations ROS, Reactive oxygen species; LEA, Late embryogenesis abundant; qRT-PCR, Quantitative real-time PCR; TBA, Thiobarbituric acid; NBT, Nitroblue tetrazolium; OE, Overexpression; WT, Wild type; MDA, Malondialdehyde; H 2 O 2 , Hydrogen peroxide; O 2 .- ; Superoxide anion; CAT, Catalase; POD, peroxidase; SOD, Superoxide dismutase; APX, Ascorbate peroxidase; ANOVA, Analysis of variance; DHN,Dehydrin; SMP, Seed maturation protein; NJ, Neighbor-Joining Declarations Declaration of c ompeting interest The authors declare no conflicts of interest related to the content of this article. Funding This work was supported by the Science and Technology Innovation Enhancement Project of Shanxi Agricultural University (CXGC2025066). Appendix A. Supplementary data Supplementary data to this article can be found online at https://-------- Author contributions Conceptualization: W.W., Z.Y., X.Z.; Investigation and writing–original draft preparation: Z.Y., Y.L.; Validation: X.Z., S.W.; Visualization: Y.Y., Y.L.; Formal analysis: W.W., Y.Y.; Resources: D.Y., L.L.; Writing–review and editing: W.W., Z.Y.; Supervision and funding acquisition: W.W. References Henry A (2013) IRRI’s drought stress research in rice with emphasis on roots: accomplishments over the last 50 years. Plant Root 7:92–106. 10.3117/plantroot.7.92 Aduse Poku S, Nkachukwu Chukwurah P, Aung HH, Nakamura I (2020) Over-Expression of a Melon Y3SK2-Type LEA Gene Confers Drought and Salt Tolerance in Transgenic Tobacco Plants. Plants (Basel) 9:1749. 10.3390/plants9121749 Ali MY, Sina AA, Khandker SS, Neesa L, Tanvir EM, Kabir A, Khalil MI, Gan SH (2020) Nutritional Composition and Bioactive Compounds in Tomatoes and Their Impact on Human Health and Disease: A Review. Foods (Basel Switzerland) 10:45. 10.3390/foods10010045 Amara I, Capellades M, Ludevid MD, Pagès M, Goday A (2013) Enhanced water stress tolerance of transgenic maize plants over-expressing LEA Rab28 gene. J Plant Physiol 170:864–873. 10.1016/j.jplph.2013.01.004 Amini S, Ghobadi C, Yamchi A (2015) Proline accumulation and osmotic stress: an overview of P5CS gene in plants. J Plant Mol Breed 3:44–55. 10.22058/jpmb.2015.17022 Banerjee A, Roychoudhury A (2016) Group II late embryogenesis abundant (LEA) proteins: structural and functional aspects in plant abiotic stress. Plant Growth Regul 79:1–17. 10.1007/s10725-015-0113-3 Bao YQ, Zeng Y, Zhao Y, Liu Y, Zhao YQ, Xiao LH, Wu ZG, Zheng YH, Jin P (2025) Phytosulfokine α treatment induces osmotic regulation to alleviate postharvest chilling injury of peach fruit by PpbHLH62. Postharvest Biol Technol 230:113746. 10.1016/j.postharvbio.2025.113746 Basu S, Kumari S, Kumar P, Kumar G, Rajwanshi R (2021) Redox imbalance impedes photosynthetic activity in rice by disrupting cellular membrane integrity and induces programmed cell death under submergence. Physiol Plant 172:1764–1778. 10.1111/ppl.13387 Battaglia M, Olvera-Carrillo Y, Garciarrubio A, Campos F, Covarrubias AA (2008) The enigmatic LEA proteins and other hydrophilins. Plant Physiol 148:6–24. 10.1104/pp.108.120725 Bindschedler LV, Minibayeva F, Gardner SL, Gerrish C, Davies DR, Bolwell GP (2001) Early signalling events in the apoplastic oxidative burst in suspension cultured French bean cells involve cAMP and Ca 2+ . New Phytol 151:185–194. 10.1046/j.1469-8137.2001.00170.x Burton-Freeman B, Sesso HD (2014) Whole food versus supplement: comparing the clinical evidence of tomato intake and lycopene supplementation on cardiovascular risk factors. Adv Nutr 5:457–485. 10.3945/an.114.005231 Celik Altunoglu Y, Baloglu P, Yer EN, Pekol S, Baloglu MC (2016) Identification and expression analysis of LEA gene family members in cucumber genome. Plant Growth Regul 80:225–241. 10.1007/s10725-016-0160-4 Chakrabortee S, Boschetti C, Walton LJ, Sarkar S, Rubinsztein DC, Tunnacliffe A (2007) Hydrophilic protein associated with desiccation tolerance exhibits broad protein stabilization function. Proc Natl Acad Sci USA 104:18073–18078. 10.1073/pnas.0706964104 Chen TZ, Zhang BL (2016) Measurements of Proline and Malondialdehyde Content and Antioxidant Enzyme Activities in Leaves of Drought Stressed Cotton. Bio-protocol 6:e1913. 10.21769/bioprotoc.1913 Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium -mediated transformation of Arabidopsis thaliana . Plant J 16:735–743. 10.1046/j.1365-313x.1998.00343.x Cui LJ, Huang Q, Yan B, Wang Y, Qian ZY, Pan JX, Kai GY (2015) Molecular cloning and expression analysis of a Cu/Zn SOD gene ( BcCSD1 ) from Brassica campestris ssp. chinensis . Food chemistry 186:306–311. 10.1016/j.foodchem.2014.07.121 Dure L 3rd, Greenway SC, Galau GA (1981) Developmental biochemistry of cottonseed embryogenesis and germination: changing messenger ribonucleic acid populations as shown by in vitro and in vivo protein synthesis. Biochemistry 20:4162–4168. 10.1021/bi00517a033 Ferdous J, Hussain SS, Shi BJ (2015) Role of microRNAs in plant drought tolerance. Plant Biotechnol J 13:293–305. 10.1111/pbi.12318 Fujita M, Fujita Y, Maruyama K, Seki M, Hiratsu K, Ohme-Takagi M, Tran LS, Yamaguchi‐Shinozaki K, Shinozaki K (2004) A dehydration‐induced NAC protein, RD26, is involved in a novel ABA‐dependent stress‐signaling pathway. Plant J 39:863–876. 10.1111/j.1365-313X.2004.02171.x Galau GA, Wang HY, Hughes DW (1993) Cotton Lea5 and Lea14 encode atypical late embryogenesis-abundant proteins. Plant Physiol 101:695–696. 10.1104/pp.101.2.695 Grelet J, Benamar A, Teyssier E, Avelange-Macherel MH, Grunwald D, Macherel D (2005) Identification in pea seed mitochondria of a late-embryogenesis abundant protein able to protect enzymes from drying. Plant Physiol 137:157–167. 10.1104/pp.104.052480 Grivna ST, Beyret E, Wang Z, Lin HF (2006) A novel class of small RNAs in mouse spermatogenic cells. Genes Dev 20:1709–1714. 10.1101/gad.1434406 Hasanuzzaman M, Fujita M (2022) Plant Oxidative Stress: Biology, Physiology and Mitigation. Plants (Basel) 11:1185. 10.3390/plants11091185 Hundertmark M, Hincha DK (2008) LEA (late embryogenesis abundant) proteins and their encoding genes in Arabidopsis thaliana . BMC Genomics 9:118. 10.1186/1471-2164-9-118 IM Petyaev (2016) Lycopene Deficiency in Ageing and Cardiovascular Disease. Oxid Med Cell Longev 2016:6. 10.1155/2016/3218605 Jia HL, Wang XM, Shi YH, Wu XM, Wang YQ, Liu JN, Fang ZH, Li CY, Dong KH (2020) Overexpression of Medicago sativa LEA4-4 can improve the salt, drought, and oxidation resistance of transgenic Arabidopsis . PLoS ONE 15:e0234085. 10.1371/journal.pone.0234085 Jisha KC, Vijayakumari K, Puthur JT (2013) Seed priming for abiotic stress tolerance: an overview. Acta Physiol Plant 35:1381–1396. 10.1007/s11738-012-1186-5 Kaur N, Sharma I, Kirat K, Pati PK (2016) Detection of Reactive Oxygen Species in Oryza sativa L.(rice). Bio-protocol 6:e2061-e2061. 10.21769/BioProtoc.2061 Kavi Kishor PB, Sreenivasulu N (2014) Is proline accumulation per se correlated with stress tolerance or is proline homeostasis a more critical issue? Plant Cell Environ 37:300–311. 10.1111/pce.12157 Kim HS, Lee JH, Kim JJ, Kim CH, Jun SS, Hong YN (2005) Molecular and functional characterization of CaLEA6 , the gene for a hydrophobic LEA protein from Capsicum annuum . Gene 344:115–123. 10.1016/j.gene.2004.09.012 Kovacs D, Kalmar E, Torok Z, Tompa P (2008) Chaperone activity of ERD10 and ERD14, two disordered stress-related plant proteins. Plant Physiol 147:381–390. 10.1104/pp.108.118208 Li C, Yan C, Sun Q, Wang J, Yuan C, Mou Y, Shan S, Zhao X (2022) Proteomic profiling of Arachis hypogaea in response to drought stress and overexpression of AhLEA2 improves drought tolerance. Plant Biol 24:75–84. 10.1111/plb.13351 Li SF, Meng HJ, Yang YF, Zhao JN, Xia YX, Wang SL, Wang F, Zheng GS, Li JB (2025) Overexpression of AtruLEA1 from Acer truncatum Bunge Enhanced Arabidopsis Drought and Salt Tolerance by Improving ROS-Scavenging Capability. Plants (Basel) 14:117. 10.3390/plants14010117 Liu Y, Wang L, Xing X, Sun LP, Pan JW, Kong XP, Zhang MY, Li DQ (2013) ZmLEA3, a multifunctional group 3 LEA protein from maize ( Zea mays L.), is involved in biotic and abiotic stresses. Plant Cell Physiol 54:944–959. 10.1093/pcp/pct047 Ma WX, Wen X, Zhao L, Dong SW, Luo D, Zhou Q, Fang LF, Liu WX, Liu ZP (2025) MsLEA_3–6 overexpression enhances drought tolerance in Medicago sativa L. BMC Plant Biol 25:1482. 10.1186/s12870-025-07101-9 Magwanga RO, Lu P, Kirungu JN, Dong Q, Hu YG, Zhou ZL, Cai XY, Wang XX, Hou YQ, Wang KB (2018) Cotton Late Embryogenesis Abundant ( LEA2 ) Genes Promote Root Growth and Confer Drought Stress Tolerance in Transgenic Arabidopsis thaliana . G3(Bethesda):2781–2803. 10.1534/g3.118.200423 Magwanga RO, Lu P, Kirungu JN, Lu HJ, Wang XX, Cai XY, Zhou ZL, Zhang ZM, Salih H, Wang KB, Liu F (2018) Characterization of the late embryogenesis abundant (LEA) proteins family and their role in drought stress tolerance in upland cotton. BMC Genet 19:6. 10.1186/s12863-017-0596-1 Nakashima K, Yamaguchi-Shinozaki K (2013) ABA signaling in stress-response and seed development. Plant Cell Rep 32:959–970. 10.1007/s00299-013-1418-1 Natarajan SK, Zhu WD, Liang XW, Zhang L, Demers AJ, Zimmerman MC, Simpson MA, Becker DF (2012) Proline dehydrogenase is essential for proline protection against hydrogen peroxide-induced cell death. Free Radic Biol Med 53:1181–1191. 10.1016/j.freeradbiomed.2012.07.002 Park SC, Kim YH, Jeong JC, Kim CY, Lee HS, Bang JW, Kwak SS (2011) Sweetpotato late embryogenesis abundant 14 ( IbLEA14 ) gene influences lignification and increases osmotic-and salt stress-tolerance of transgenic calli. Planta 233:621–634. 10.1007/s00425-010-1326-3 Perveen R, Suleria HAR, Anjum FM, Butt MS, Pasha I, Ahmad S (2015) Tomato ( Solanum lycopersicum ) Carotenoids and Lycopenes Chemistry; Metabolism, Absorption, Nutrition, and Allied Health Claims—A Comprehensive Review. Crit Rev Food Sci Nutr 55:919–929. 10.1080/10408398.2012.657809 Reyes JL, Campos F, Wei H, Arora R, Yang Y, Karlson DT, Covarrubias AA (2008) Functional dissection of hydrophilins during in vitro freeze protection. Plant Cell Environ 31:1781–1790. 10.1111/j.1365-3040.2008.01879.x Rodríguez-Valentín R, Battaglia M, Campos F, Solórzano RM, Rosales MA, Covarrubias AA (2014) Group 6 Late Embryogenesis Abundant (LEA) Proteins in Monocotyledonous Plants: Genomic Organization and Transcript Accumulation Patterns in Response to Stress in Oryza sativa . Plant Mol Biology Report 32:198–208. 10.1007/s11105-013-0641-9 Sharma A, Kumar D, Kumar S, Rampuria S, Reddy AR, Kirti PB (2016) Ectopic Expression of an Atypical Hydrophobic Group 5 LEA Protein from Wild Peanut, Arachis diogoi Confers Abiotic Stress Tolerance in Tobacco. PLoS ONE 11:e0150609. 10.1371/journal.pone.0150609 Sofo A, Scopa A, Nuzzaci M, Vitti A (2015) Ascorbate Peroxidase and Catalase Activities and Their Genetic Regulation in Plants Subjected to Drought and Salinity Stresses. Int J Mol Sci 16:13561–13578. 10.3390/ijms160613561 Tolleter D, Jaquinod M, Mangavel C, Passirani C, Saulnier P, Manon S, eyssier E, Payet N, Avelange-Macherel MH, Macherel D (2007) Structure and function of a mitochondrial late embryogenesis abundant protein are revealed by desiccation. Plant Cell 19:1580–1589. 10.1105/tpc.107.050104 Waadt R, Seller CA, Hsu PK, Takahashi Y, Munemasa S, Schroeder JI (2022) Plant hormone regulation of abiotic stress responses. Nat Rev Mol Cell Biol 23:680–694. 10.1038/s41580-022-00479-6 Wang HQ, Wu YC, Yang XB, Guo XR, Cao XY (2017) SmLEA2 , a gene for late embryogenesis abundant protein isolated from Salvia miltiorrhiza , confers tolerance to drought and salt stress in Escherichia coli and S. miltiorrhiza . Protoplasma 254:685–696. 10.1007/s00709-016-0981-z Wang MZ, Li P, Li C, Pan YL, Jiang XY, Zhu DY, Zhao Q, Yu JJ (2014) SiLEA14, a novel atypical LEA protein, confers abiotic stress resistance in foxtail millet. BMC Plant Biol 14:290. 10.1186/s12870-014-0290-7 Wang Q, Lei XJ, Wang YH, Di P, Meng XR, Peng WY, Rong JB, Wang YP (2024) Genome-wide identification of the LEA gene family in Panax ginseng : Evidence for the role of PgLEA2-50 in plant abiotic stress response. Plant Physiol Biochem 212:108742. 10.1016/j.plaphy.2024.108742 Wang WB, Kim YH, Lee HS, Kim KY, Deng XP, Kwak SS (2009) Analysis of antioxidant enzyme activity during germination of alfalfa under salt and drought stresses. Plant Physiol Biochem 47:570–577. 10.1016/j.plaphy.2009.02.009 Yamaguchi-Shinozaki K, Urao S, Koizumi M, Shinozaki K (1992) Molecular Cloning and Characterization of 9 cDNAs for Genes That Are Responsive to Desiccation in Arabidopsis thaliana : SequenceAnalysis of One cDNA Clone That Encodes a Putative Transmembrane Channel Protein. Plant Cell Physiol 33:217–224. 10.1093/oxfordjournals.pcp.a078243 Yu J, Lai YM, Wu X, Wu G, Guo CK (2016) Overexpression of OsEm1 encoding a group I LEA protein confers enhanced drought tolerance in rice. Biochem Biophys Res Commun 478:703–709. 10.1016/j.bbrc.2016.08.010 Zaman Khan N, Lal S, Ali W, Aasim M, Mumtaz S, Kamil A, Shad Bibi N (2020) Distribution and Classification of Dehydrins in Selected Plant Species Using Bioinformatics Approach. Iran J Biotechnol 18:e2680. 10.30498/IJB.2020.2680 Zheng JX, Su HX, Lin RY, Zhang H, Xia KF, Jian SG, Zhang M (2019) Isolation and characterization of an atypical LEA gene ( IpLEA ) from Ipomoea pes-caprae conferring salt/drought and oxidative stress tolerance. Sci Rep 9:14838. 10.1038/s41598-019-50813-w Supplementary Files SupplementaryTable.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Major revision 16 Mar, 2026 Reviewers agreed at journal 25 Feb, 2026 Reviewers invited by journal 10 Feb, 2026 Editor assigned by journal 09 Feb, 2026 First submitted to journal 05 Feb, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8801747","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":589225842,"identity":"f5118b99-0862-4ed3-9bd8-c32ec496ca87","order_by":0,"name":"Zhehua Yan","email":"","orcid":"","institution":"Shanxi Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Zhehua","middleName":"","lastName":"Yan","suffix":""},{"id":589225843,"identity":"a38b4df0-64fd-4049-85ee-c0dc20639826","order_by":1,"name":"Yu Lei","email":"","orcid":"","institution":"Shanxi Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Lei","suffix":""},{"id":589225844,"identity":"a5188d89-e53d-4af4-84a4-236f534f6b60","order_by":2,"name":"Xuan Zou","email":"","orcid":"","institution":"Shanxi Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Xuan","middleName":"","lastName":"Zou","suffix":""},{"id":589225845,"identity":"b5355d95-6d7a-4625-b042-74978de16630","order_by":3,"name":"Sijie Wang","email":"","orcid":"","institution":"Shanxi Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Sijie","middleName":"","lastName":"Wang","suffix":""},{"id":589225846,"identity":"ab4a78d6-63b5-4e29-829b-3484ca9d54e8","order_by":4,"name":"Yanxin Yang","email":"","orcid":"","institution":"Shanxi Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yanxin","middleName":"","lastName":"Yang","suffix":""},{"id":589225847,"identity":"92d645c0-4784-45b8-8024-6e863db5bf73","order_by":5,"name":"Dongjing Yang","email":"","orcid":"","institution":"Xuzhou Institute of Agricultural Sciences in Jiangsu Xuhuai Distric: Chinese Academy of Agricultural Sciences Sweet Potato Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Dongjing","middleName":"","lastName":"Yang","suffix":""},{"id":589225848,"identity":"01f85884-8564-46f8-8209-1c826b2e64a0","order_by":6,"name":"Lingzhi Li","email":"","orcid":"","institution":"Shanxi Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Lingzhi","middleName":"","lastName":"Li","suffix":""},{"id":589225849,"identity":"4858d54b-c7be-4cce-8823-dcbe5af3aa10","order_by":7,"name":"Wenbin Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAArklEQVRIiWNgGAWjYJACCQaGA3Js7M0HSNNizMdzLIE0LYnzJHIUiFMuPyP34G3etjvpbQw5DAw/KrYR1mJw5lyyNW/bs9w2hrMHGHvO3CZCC3uPmXTutsO5bYx9CcyMbURokW/mAWtJZ2PmMSBOC8NxiC0JbGzEajE4c8bY+u+/Z4ZtPGwJB4nyi/yMHMObM87ckZef//jggx8VxDgMGRwgUf0oGAWjYBSMAlwAAAZzPCutokfbAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-5289-4489","institution":"Shanxi Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Wenbin","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2026-02-06 02:09:49","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8801747/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8801747/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102757519,"identity":"9e6c5bec-c02b-444f-870b-966a10273e14","added_by":"auto","created_at":"2026-02-16 10:00:10","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1280895,"visible":true,"origin":"","legend":"\u003cp\u003eSequence alignment and phylogenetic analysis of SlLEA_2-26 homologous proteins. (a) Phylogenetic analysis of SlLEA_2-26 homologous proteins. SlLEA_2-26 is indicated by a black triangle. (b) Multiple sequence alignment of SlLEA_2-26 homologous proteins. Different color blocks represent sequence similarity, where black, pink, and blue indicate 100%, 75%, and 50% sequence similarity, respectively. The red underlined region denotes the LEA_2 conserved domain\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-8801747/v1/8cae0f9aa2664922fc2816da.png"},{"id":102757518,"identity":"e1cc5db1-93c8-4728-8cea-b22e4c999e64","added_by":"auto","created_at":"2026-02-16 10:00:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":89312,"visible":true,"origin":"","legend":"\u003cp\u003eExpression patterns of the \u003cem\u003eSlLEA_2-26\u003c/em\u003e gene in different tissues and under abiotic stress conditions. (a) Tissue-specific expression pattern of \u003cem\u003eSlLEA_2-26\u003c/em\u003e. G0: Dry, non-germinated seeds; G4: Seeds germinated for 4 days; C: Cotyledons; H: Hypocotyls; R: Roots; S: Stems; L: Leaves; Fl: Flowers; Fr: Fruits. (b) Expression pattern of \u003cem\u003eSlLEA_2-26\u003c/em\u003e under different stresses. 15% PEG6000, 200 mM NaCl, 44℃, 4℃, 800 μM CuSO\u003csub\u003e4\u003c/sub\u003e, 5 mM Pb(CH\u003csub\u003e3\u003c/sub\u003eCOO)\u003csub\u003e2\u003c/sub\u003e, 800 μM CdSO\u003csub\u003e4\u003c/sub\u003e, and 75 μM ABA were added to 1/2 Hoagland nutrient solution, respectively. Significant differences (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01) are denoted by different lowercase letters; Asterisks indicate significant differences versus the 0 h time point (*\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05 and **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01)\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-8801747/v1/2e135c62b9d3d932862ed81b.png"},{"id":102757535,"identity":"92ed5f2b-e240-4a8d-9c8d-e9d114e240be","added_by":"auto","created_at":"2026-02-16 10:00:12","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":381641,"visible":true,"origin":"","legend":"\u003cp\u003eSeed germination and root length assays in Arabidopsis WT and \u003cem\u003eSlLEA_2-26\u003c/em\u003e overexpression lines under 200 mM mannitol and 100 mM NaCl treatments. (a) Seed germination phenotypes. (b) Seed germination rate. (c) Seedling root elongation phenotypes. (d) Fresh weight. (e) Root length. Significant differences between the OE lines and the WT are indicated by asterisks (*\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05 and **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01)\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-8801747/v1/0f1f3c10e5a5ccaa0ec62999.png"},{"id":102757520,"identity":"a0115763-d7c8-4713-ba16-e0b138abb0f6","added_by":"auto","created_at":"2026-02-16 10:00:10","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":649203,"visible":true,"origin":"","legend":"\u003cp\u003ePhenotypic and physiological characteristic analysis of transgenic Arabidopsis under drought stress. (a) Phenotypes of OEs and WT at 0, 7, 14 days of drought stress and 3 days after rewatering. The contents of soluble sugar (b), proline (c), MDA (h), H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (i), and O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e.-\u003c/sup\u003e (j). The activities of SOD (d), POD (e), APX (f), and CAT (g) after drought and followed by rewatering. Significant differences between the OE lines and the WT are indicated by asterisks (*\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05 and **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01)\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-8801747/v1/fcbde264094a341f45466c6f.png"},{"id":102757512,"identity":"d9d87e97-cdee-4c85-9b66-b9a78a7d160b","added_by":"auto","created_at":"2026-02-16 10:00:08","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":166955,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of \u003cem\u003eSlLEA_2-26\u003c/em\u003eoverexpression on the expression of stress-responsive genes in Arabidopsis under drought stress. mRNA levels of \u003cem\u003eSlLEA_2-26\u003c/em\u003e (a), \u003cem\u003eAtP5CS1\u003c/em\u003e (b), \u003cem\u003eAtCSD1\u003c/em\u003e (c), \u003cem\u003eAtNCED3\u003c/em\u003e (d), \u003cem\u003eAtRD26\u003c/em\u003e (e), and \u003cem\u003eAtRD29A\u003c/em\u003e(f) in OEs and WT under drought stress. Significant differences between the OE lines and the WT are indicated by asterisks (*\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05 and **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01)\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-8801747/v1/a8829e02c1be5bc5d6bb66d1.png"},{"id":102757523,"identity":"0d1b5662-b80b-4379-b486-850a454a01fd","added_by":"auto","created_at":"2026-02-16 10:00:11","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":652106,"visible":true,"origin":"","legend":"\u003cp\u003ePhenotypic and physiological characteristic analysis in transgenic Arabidopsis under salt stress. (a) Phenotypes of OEs and WT under 15 days of salt stress. The contents of soluble sugar (b), proline (c), MDA (h), H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (i), and O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e.-\u003c/sup\u003e (j). The activities of SOD (d), POD (e), APX (f), and CAT (g) after 15 days of salt stress. Significant differences between the OE lines and the WT are indicated by asterisks (*\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05 and **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01)\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-8801747/v1/716c1079003c2cb56a6545a5.png"},{"id":102757515,"identity":"e584167b-6a36-457c-8a8a-e39e49090156","added_by":"auto","created_at":"2026-02-16 10:00:09","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":181189,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of \u003cem\u003eSlLEA_2-26\u003c/em\u003eoverexpression on the expression of stress-responsive genes in Arabidopsis under salt stress. mRNA levels of \u003cem\u003eSlLEA_2-26\u003c/em\u003e (a), \u003cem\u003eAtP5CS1\u003c/em\u003e (b), \u003cem\u003eAtCSD1\u003c/em\u003e(c), \u003cem\u003eAtNCED3\u003c/em\u003e (d), \u003cem\u003eAtRD26\u003c/em\u003e (e), and \u003cem\u003eAtRD29A\u003c/em\u003e (f) in OEs and WT under salt stress. Significant differences between the OE lines and the WT are indicated by asterisks (*\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05 and **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01)\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-8801747/v1/b7e81e1c558441e69fcce950.png"},{"id":102757571,"identity":"c235b9ee-07e1-4d7d-9159-ac52a97ca3bf","added_by":"auto","created_at":"2026-02-16 10:00:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4194767,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8801747/v1/ac700e34-3028-4046-ad03-3047c80bef47.pdf"},{"id":102757516,"identity":"cdf002ca-8986-4919-bd04-1c73938e797b","added_by":"auto","created_at":"2026-02-16 10:00:09","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":16880,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable.docx","url":"https://assets-eu.researchsquare.com/files/rs-8801747/v1/33762457413d1f8bcf54ec27.docx"}],"financialInterests":"","formattedTitle":"Overexpression of the tomato SlLEA_2-26 gene enhances the tolerance to drought and salt stresses in Arabidopsis thaliana","fulltext":[{"header":"Headings","content":"\u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003e \u003cem\u003eSlLEA_2\u0026ndash;26\u003c/em\u003e gene was cloned from the tomato cultivar 1436.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eThe multiple environmental stresses significantly increased the expression of \u003cem\u003eSlLEA_2\u0026ndash;26\u003c/em\u003e gene in tomato leaves.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eOverexpression of \u003cem\u003eSlLEA_2\u0026ndash;26\u003c/em\u003e significantly improved the salt and drought stress tolerance in transgenic Arabidopsis lines.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e"},{"header":"1. Introduction","content":"\u003cp\u003eTomato (\u003cem\u003eSolanum lycopersicum\u003c/em\u003e L.) is a globally cultivated crop of high nutritional value (Ali MY et al. 2020). It serves as the richest dietary source of lycopene, a carotenoid with an antioxidant capacity 100 times that of vitamin E (Burton-Freeman B and Sesso HD 2014). Lycopene is associated with delayed cellular aging and a reduced risk of certain cancers and cardiovascular diseases (Petyaev IM 2016; Perveen R et al. 2015). Furthermore, tomatine present in tomatoes exhibits antibacterial and anti-inflammatory properties. However, drought and salinization constitute major environmental constraints that impede crop growth, significantly reduce crop yields, and limit agricultural and economic development.\u003c/p\u003e\n\u003cp\u003eLate embryogenesis abundant (LEA) proteins, widely distributed in plants, accumulate abundantly during the late stages of embryo development (Banerjee A and Roychoudhury A 2016; Dure L 3rd et al. 1981). These proteins can chelate metal ions (Grelet J et al. 2005), protect enzyme activity (Reyes JL et al. 2008), and mitigate cellular damage under drought stress (Tolleter D et al. 2007). Initially isolated from \u003cem\u003eGossypium hirsutum\u003c/em\u003e cotyledons during embryogenesis, LEA proteins have since been studied in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e (Hundertmark M and Hincha DK 2008), \u003cem\u003eOryza sativa\u003c/em\u003e (Rodríguez-Valentín R et al. 2014), \u003cem\u003eCucumis sativus\u003c/em\u003e (Celik Altunoglu Y et al. 2016), and \u003cem\u003eZea mays\u003c/em\u003e (Liu Y et al. 2013). By shielding plant cells from dehydration-induced damage caused by drought and salinity, LEA proteins play a critical role in sustaining normal plant growth and development (Zaman Khan N et al. 2020).\u003c/p\u003e\n\u003cp\u003eAs the LEA protein family continues to expand, its classification framework has been progressively refined. Currently, LEA proteins are primarily categorized into eight subgroups, including dehydrins (DHN), seed maturation proteins (SMP), and LEA_1 through LEA_6. Most of these proteins are characterized by high hydrophilicity. A notable exception is the LEA_2 subgroup, which is enriched in hydrophobic residues, rendering it the least hydrophilic member of the family. According to an earlier classification proposed by Battaglia’s group, LEA proteins were divided into seven groups (groups 1–7). Among these, groups 1, 2, 3, 4, 6, and 7 are considered typical LEA proteins due to their marked hydrophilicity and conserved motifs. In contrast, group 5 is classified as atypical because of its low sequence homology and significantly higher proportion of hydrophobic amino acids. Notably, LEA_2 was originally assigned to group 5 in this scheme (Battaglia M et al. 2008), its established crucial role in plant abiotic stress responses (Galau GA et al. 1993).\u003c/p\u003e\n\u003cp\u003eResearch indicates that nearly all LEA gene promoters contain cis-regulatory elements such as ABRE, MBS, W-box, and TAC, which are integral to the plant stress-response network (Magwanga RO et al. 2018). Moreover, miRNA-mediated reprogramming of gene expression serves as a key defense mechanism under drought stress (Ferdous J et al. 2015). Functioning primarily at the post-transcriptional level, numerous miRNAs regulate plant adaptation to abiotic stresses (Grivna ST et al. 2006). For instance, in cotton, 48 distinct miRNAs are predicted to target 63 LEA_2 genes, accounting for approximately 40% of this subfamily, with several genes being targeted by more than six miRNAs. These miRNAs also regulate multiple known drought-responsive genes and target stress-related cis-elements, including ABRE, DRE/CRT, MYBS, and LTRE (Magwanga RO et al. 2018). Through this coordinated regulation of stress-associated genes and regulatory motifs, miRNAs play a vital role in plant responses to drought and salinity. This positions LEA_2 as a pivotal integrator of stress signals within the regulatory network, acting as both a key effector and a synergistic regulator in plant abiotic stress defense.\u003c/p\u003e\n\u003cp\u003eLEA_2 proteins function as crucial molecular components in plant responses to abiotic stress, and their roles have been extensively characterized. Genes encoding LEA_2 proteins, identified across diverse species, are consistently involved in regulating plant adaptation to drought and high salinity. For example, the expression of \u003cem\u003eCaLEA6\u003c/em\u003e in pepper and \u003cem\u003eAdLEA\u003c/em\u003e in peanut is significantly upregulated in response to NaCl and PEG treatments, indicating their active participation in coping with salt-induced and drought-induced water deficit (Kim HS et al. 2005; Sharma A et al. 2016). Furthermore, heterologous expression of \u003cem\u003eSmLEA2\u003c/em\u003e from \u003cem\u003eSalvia miltiorrhiza\u003c/em\u003e enhances \u003cem\u003eEscherichia coli\u003c/em\u003e growth under salt and drought stress. Similarly, transgenic plants overexpressing this gene show increased stress tolerance, whereas its suppression leads to greater susceptibility (Wang HQ et al. 2017). Notably, maize Rab28 is classified within group 5 alongside LEA_2 proteins. Overexpression of the \u003cem\u003eRab28\u003c/em\u003e gene significantly improves osmotic stress tolerance in maize (Amara I et al. 2013).\u003c/p\u003e\n\u003cp\u003eMechanistically, LEA_2 proteins enhance stress resistance by modulating stress-related signaling pathways. For instance, in transgenic Arabidopsis overexpressing the cotton gene \u003cem\u003eCotAD_24498\u003c/em\u003e, drought stress significantly upregulates the expression of \u003cem\u003eABF4\u003c/em\u003e and \u003cem\u003eRD29A\u003c/em\u003e (Magwanga RO et al. 2018). The transcription factor ABF4, a central component of the ABA signaling pathway, activates a suite of drought-responsive genes by binding to ABRE cis-elements within their promoters. Concurrently, the elevated expression of \u003cem\u003eRD29A\u003c/em\u003e, a well-established drought stress marker, directly reflects a heightened cellular capacity to respond to water deficit. Thus, \u003cem\u003eCotAD_24498\u003c/em\u003e likely strengthens drought tolerance by constitutively activating the ABA signaling network and its downstream targets. In addition, under drought stress, tobacco plants overexpressing the peanut \u003cem\u003eAdLEA\u003c/em\u003e gene significantly upregulate the expression of the \u003cem\u003eNtERD10c\u003c/em\u003e gene. \u003cem\u003eERD10c\u003c/em\u003e encodes a hydrophilic group 2 LEA protein, which contributes to dehydration tolerance by binding water molecules, stabilizing labile enzymes, and protecting macromolecular structures (Kovacs D et al. 2008). Transgenic plants may synthesize increased amounts of protective molecular chaperones, thereby more effectively mitigating water loss under dehydration or drought stress (Chakrabortee S et al. 2007).\u003c/p\u003e\n\u003cp\u003eAlthough the overexpression of \u003cem\u003eLEA_2\u003c/em\u003e in plants can enhance stress tolerance, studies on the function of tomato \u003cem\u003eLEA_2\u003c/em\u003e genes in response to abiotic stress are still relatively limited. In this study, a tomato \u003cem\u003eSlLEA_2-26\u003c/em\u003e gene was cloned from cultivar 1436, and transgenic Arabidopsis overexpression lines were constructed. Additionally, its expression profiles were analyzed across different tomato tissues and under various stress conditions. The tolerance of transgenic Arabidopsis to salt and drought stresses was assessed, and the expression levels of stress-related genes were measured. These results elucidated the molecular mechanism by which \u003cem\u003eSlLEA_2-26\u003c/em\u003e responds to abiotic stress, provided a theoretical basis and genetic resources for stress-resistant breeding in tomatoes and other crops.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cp\u003e\u003cem\u003e2.1. Plant materials and growing conditions\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eArabidopsis thaliana\u003c/em\u003e ecotype Colombia-0 (Col-0) and tomato cultivar 1436 were employed as wild types (WT). Various tomato tissues, including seeds, cotyledons, hypocotyls, roots, stems, leaves, flowers, and fruits, were collected from plants grown under normal conditions for tissue-specific expression profiling. To assess mRNA expression levels of the \u003cem\u003eSlLEA_2-26\u003c/em\u003e gene under different abiotic stresses, 3-week-old tomato plants were grown in 1/2 Hoagland nutrient solution supplemented with 200 mM NaCl, 15% PEG6000, 800 μM CdSO\u003csub\u003e4\u003c/sub\u003e, 800 μM CuSO\u003csub\u003e4\u003c/sub\u003e, 5 mM Pb(CH\u003csub\u003e3\u003c/sub\u003eCOO)\u003csub\u003e2\u003c/sub\u003e, 75 μM ABA, respectively, or subjected to 44 ℃ and 4 ℃ treatments. At 0, 1, 3, 6, 12, 24, and 48 h, the third and fourth leaves from the bottom were collected as experimental material for further quantitative real-time PCR analysis.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.2. Phylogenetic and sequence alignment analysis of SlLEA_2-26\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eHomologous protein sequences were retrieved using the NCBI-BLAST tool. Multiple sequence alignment of the protein sequences was performed with DNAMAN 5.2.2.0 software. A phylogenetic tree was constructed with MEGA 7.1 software based on the Neighbor-Joining (NJ) method. The subcellular localization of the SlLEA_2-26 protein was predicted using WoLF PSORT (https://wolfpsort.hgc.jp/).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.3. Quantitative real-time PCR (qRT-PCR) analysis of gene expression\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted using TriQuick reagent (Solarbio, China), and cDNA was synthesized with a reverse transcription kit (Biosharp, Anhui, China) for subsequent qRT-PCR analysis. The specific primers designed via Primer-BLAST on the NCBI website are provided in Table S1. The reagent used in the qRT-PCR reaction was 2×M5 HiPer SYBR Premix EsTaq (with Tli RNaseH). \u003cem\u003eSlActin\u003c/em\u003e was used as the internal control gene for qRT-PCR analysis. The relative expression levels of \u003cem\u003eSlLEA_2-26\u003c/em\u003e were calculated using the 2\u003csup\u003e-\u003c/sup\u003e\u003csup\u003e△△\u003c/sup\u003e\u003csup\u003eCT\u003c/sup\u003e method.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.4. Generation of SlLEA_2-26- overexpression lines in Arabidopsis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe full-length \u003cem\u003eSlLEA_2-26\u003c/em\u003e gene was inserted into the pCAMBIA1302 driven by a stress-inducible promoter \u003cem\u003eSWPA2\u003c/em\u003e to generate the recombinant vector pCAMBIA1302-\u003cem\u003eSlLEA_2-26\u003c/em\u003e. The construct was transformed into \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e GV3101 for Col-0 transformation via floral dipping (Clough SJ and Bent AF 1998). T\u003csub\u003e3\u003c/sub\u003e homozygous overexpression lines were selected via hygromycin resistance (50 mg·L\u003csup\u003e-1\u003c/sup\u003e) and verified through genomic PCR. Three resulting T\u003csub\u003e3\u003c/sub\u003e transgenic lines (OE6, OE9, OE11) were randomly selected for subsequent functional analysis.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.5. Abiotic stress tolerance assay in T\u003csub\u003e3\u003c/sub\u003e transgenic Arabidopsis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eFor germination assays, seeds of both WT and OEs were cultured on 1/2 MS solid medium under osmotic and salt stress conditions, imposed by supplementation with 200 mM mannitol or 100 mM NaCl, respectively. After 7 days of incubation, the germination rates were calculated (germination was defined as the appearance of the radicle). For root growth tests, 5-day-old seedlings with a similar root length were transferred to 1/2 MS solid medium supplemented with 225 mM mannitol and 125 mM NaCl, respectively. The root lengths of the vertically cultured seedlings were measured after 7 days of growth.\u003c/p\u003e\n\u003cp\u003eTo assess the potential of \u003cem\u003eSlLEA_2-26\u003c/em\u003e overexpression in improving salt and drought tolerance at the whole plant level, 20-day-old uniform seedlings of WT and OEs plants were withheld from irrigation 14 days and then rewatered for 3 day, or subjected to irrigation with 225 mM NaCl solution for 15 days. Leaves were collected at 0, 7, 14 days after irrigation cessation, and 3 days after resumption. Under salt stress, leaves were sampled at 0, 5, 10, 15 days for further analysis.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.6. Determination of MDA, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e·-\u003c/sup\u003e, soluble sugar and proline content\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe Thio-barbituric acid (TBA) method was used to measure malondialdehyde (MDA) content (Wang WB et al. 2009). Evaluation of plant hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) content was performed by the xylenol orange method (Bindschedler LV et al. 2001). The superoxide anion (O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e·-\u003c/sup\u003e) content was determined according to the hydroxylamine oxidation method (Kaur N et al. 2016). Soluble sugar and proline contents were determined using corresponding assay Kits (Sangon Biotech, Shanghai, China).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.7.Measurement of antioxidant enzyme activity\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe activities of superoxide dismutase (SOD) and peroxidase (POD) were determined using the nitroblue tetrazolium (NBT) photochemical reduction and guaiacol methods, respectively (Chen TZ and Zhang BL 2016). SOD activity was quantified as the amount of enzyme causing 50% inhibition of NBT reduction. Catalase (CAT) and ascorbate peroxidase (APX) activities were assayed using spectrophotometric methods as previously reported (Wang WB et al. 2009). The units of all antioxidant enzymes activity were represented as U·mg \u003csup\u003e-1\u003c/sup\u003e protein.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.8. Determination of relevant gene expression levels\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eRNA was extracted from stress-treated samples using TriQuick reagent (Solarbio, China), and cDNA was synthesized using a reverse transcription kit (Biosharp, Anhui, China). The expression levels of \u003cem\u003eSlLEA_2-26\u003c/em\u003e, \u003cem\u003eAtP5CS1\u003c/em\u003e, \u003cem\u003eAtCSD1\u003c/em\u003e, \u003cem\u003eAtNCED3\u003c/em\u003e, \u003cem\u003eAtRD26\u003c/em\u003e, and \u003cem\u003eAtRD29A\u003c/em\u003e were determined by qRT-PCR using AtActin genes as internal reference gene. The qRT-PCR primers were listed in Supplementary Table S1.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.9. Statistical analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAll statistical analyses in this study were performed using IBM SPSS 25.0 (IBM Corp, USA). Data represent mean\u0026nbsp;±\u0026nbsp;SE of three biological replicates, and statistical differences were assessed using analysis of variance (ANOVA) and independent samples \u003cem\u003et\u003c/em\u003e-test. The significant differences at \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 and extremely significant differences at \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 between WT and OE Arabidopsis lines were determined (\u003cem\u003e*P\u0026nbsp;\u003c/em\u003e\u0026lt; 0.05 and \u003cem\u003e**P\u0026nbsp;\u003c/em\u003e\u0026lt; 0.01).\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cem\u003e3.1. Cloning and sequence analysis of SlLEA_2-26 gene\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eIn our previous study, the tomato \u003cem\u003eLEA_2\u003c/em\u003e gene family was systematically analyzed. Based on its chromosomal location, the gene investigated in this work was identified and named (data not shown). The \u003cem\u003eSlLEA_2-26\u003c/em\u003e cDNA was isolated from tomato cultivar 1436 (GenBank Accession No. PP130721). This gene has a full-length of 666 bp encoding 221 amino acids in which contains abundant 27 leucine residues, while tryptophan is present only once. The SlLEA_2-26 protein possesses a transmembrane structure and is predicted as an unstable, basic, and hydrophobic protein. WoLF PSORT analysis predicted a cytoplasmic localization for SlLEA_2-26. Tomato SlLEA_2-26 was the most closely related to the LEA homologs of \u003cem\u003eQuillaja saponaria\u003c/em\u003e through phylogenetic analysis (Fig. 1a). Multiple sequence alignment indicated that SlLEA_2-26 protein contains the typical LEA_2 domain (Fig. 1b). Taken together, these results suggested that SlLEA_2-26 is a functional LEA_2 protein.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.2. Tissue-specific and stress-induced expression profiles analysis of SlLEA_2-26 in tomato\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eqRT-PCR analysis measured \u003cem\u003eSlLEA_2-26\u003c/em\u003e expression in seeds, cotyledons, hypocotyls, roots, stems, leaves, flowers, and fruits. The results indicated that the \u003cem\u003eSlLEA_2-26\u003c/em\u003e gene is differentially expressed across various tissues, with significantly higher expression levels predominantly observed in flowers and fruits. In contrast, the relative expression of \u003cem\u003eSlLEA_2-26\u003c/em\u003e was found to be minimal in seeds at post-germination 4 days (Fig. 2a).\u003c/p\u003e\n\u003cp\u003eTo elucidate the response pattern of \u003cem\u003eSlLEA_2-26\u003c/em\u003e to various stresses, its expression dynamics under treatments of NaCl, PEG, ABA, extreme temperatures, and four metal ions were further analyzed by qRT-PCR (Fig. 2b). Under eight different abiotic stresses, the expression levels of \u003cem\u003eSlLEA_2-26\u003c/em\u003e generally showed an initial increase followed by a decrease over time. The gene exhibited strong responses to PEG, NaCl, copper, and lead stresses, and lower responses to extreme temperatures, cadmium, and ABA stresses. The \u003cem\u003eSlLEA_2-26\u003c/em\u003e gene could be upregulated and reached a peak at 24 h under drought stress, while these peaks occurred at 6 h and 3 h under copper and lead stress, respectively. However, \u003cem\u003eSlLEA_2-26\u003c/em\u003e expression exhibited a rapid but short-lived response to lead stress, primarily functioning during the early stages of stress exposure. Additionally, under NaCl stress, the relative expression level of the \u003cem\u003eSlLEA_2-26\u003c/em\u003e sharply increased to a peak at 1 h, followed by an overall downward trend while remaining significantly higher than that before the treatment. This result indicated that the \u003cem\u003eSlLEA_2-26\u003c/em\u003e exhibits a rapid and sustained response to salt stress.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn summary, the induced expression of the \u003cem\u003eSlLEA_2-26\u003c/em\u003e gene exhibited a tissue specificity. The different expression profile under the various stress suggested that the \u003cem\u003eSlLEA_2-26\u003c/em\u003e gene can rapid or sustained response to adverse environment.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.3. Overexpression of the SlLEA_2-26 enhances seed germination rate and root elongation under drought and salt stress\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThree homozygous \u003cem\u003eSlLEA_2-26\u003c/em\u003e-overexpressing Arabidopsis lines (OE6, OE9, and OE11) verified by genomic PCR were selected to investigate whether overexpression of \u003cem\u003eSlLEA_2-26\u003c/em\u003e can affect seed germination. Under normal growth conditions, seed germination rates of WT and \u003cem\u003eSlLEA_2-26\u003c/em\u003e overexpressing lines were consistent with their phenotypes and showed no significant differences (Fig. 3a,b). However, the OEs exhibited significantly higher germination rates than that of WT under mannitol and NaCl stresses. For instance, the germination rate under NaCl stress was approximately 88% for OEs seeds, in contrast to 72% for WT.\u003c/p\u003e\n\u003cp\u003eThe root system of plants is a key organ to acquire nutrients from the external environment, and the stress tolerance of Arabidopsis at the seedling stage can be evaluated by root elongation under adverse environment (Jisha KC et al. 2013). No significant difference in growth phenotype (Fig. 3c), fresh weight (Fig. 3d), or root length (Fig. 3e) was detected between WT and OEs seedlings under normal growth conditions. In contrast, under osmotic stress induced by mannitol and ionic stress imposed by NaCl, OEs seedlings demonstrated significantly enhanced root elongation and increased fresh weight, respectively, relative to WT.\u003c/p\u003e\n\u003cp\u003eIn conclusion, heterologous overexpression of \u003cem\u003eSlLEA_2-26\u003c/em\u003e gene significantly promoted seed germination and root elongation in Arabidopsis under drought and salt stress, and contributed to maintaining higher fresh weight in OE plants.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.4. Overexpression of SlLEA_2-26 enhances drought stress tolerance in adult Arabidopsis plants\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe effects of \u003cem\u003eSlLEA_2-26\u003c/em\u003e overexpression on drought tolerance in adult Arabidopsis plants were further investigated through potted-plant water control experiment. Under normal growth conditions WT and Arabidopsis plants exhibited comparable growth phenotypes. During drought stress, however, WT plants exhibited leaves purpling, and overall darkening, while a portion of leaves in OEs remained green. Three days after rewatering, WT plants showed more severe wilting and reduction in plant size compared to the OE lines (Fig. 4a).\u003c/p\u003e\n\u003cp\u003eProline and soluble sugars, as typical osmoregulatory substances, are important indicators for evaluating stress resistance. Their accumulation under stress conditions helps maintain cellular osmotic pressure and alleviates osmotic damage (Waadt R et al. 2022). Before treatment, soluble sugar and proline contents showed no significant differences between WT and OE Arabidopsis plants (Fig. 4b,c). As drought treatment duration extension, soluble sugar and proline contents in the OEs gradually increased and remained significantly higher than those in WT. At drought 14\u003csup\u003eth\u003c/sup\u003e day, soluble sugar content in lines OE6, OE9, and OE11 reached levels as high as 37.28, 37.05, and 34.11 mg·g \u003csup\u003e-1\u003c/sup\u003e FW, respectively (Fig. 4b). After rewatering 3 days, the contents of these osmoregulatory substances decreased, while those in OEs lines still exhibited significantly higher levels than WT (Fig. 4b,c). The results indicated that \u003cem\u003eSlLEA_2-26\u003c/em\u003e-overexpression in Arabidopsis improves the drought stress tolerance by promoting the accumulation of osmotic regulatory substances.\u003c/p\u003e\n\u003cp\u003eDrought and salinity stress disrupted cellular redox balance, which led to the accumulation of reactive oxygen species (ROS) and thereby induced oxidative damage in plants (Li SF et al. 2025). During drought and rehydration periods, the MDA, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e·-\u003c/sup\u003e levels in transgenic Arabidopsis were significantly lower than those in WT plants (Fig. 4h-j). The difference became more significant after 14 days of drought, with MDA content in OE Arabidopsis plants reduced by approximately 36% relative to WT. (Fig. 4h). Additionally, all the SOD, POD, APX, and CAT enzyme activities in the OE lines gradually increased and were significantly higher than those in WT under drought stress (Fig. 4d-g). Specifically, SOD activity in lines OE6, OE9, and OE11 was 1.52-, 1.43-, and 1.5-fold higher than in WT at drought 14\u003csup\u003eth\u003c/sup\u003e day, respectively (Fig. 4d).\u003c/p\u003e\n\u003cp\u003eThese findings suggested that overexpression of \u003cem\u003eSlLEA_2-26\u003c/em\u003e gene in OEs improves the antioxidant enzymes activity, reduces ROS accumulation and membrane lipid peroxidation damage, and ultimately enhances the stress tolerance to drought.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.5. Drought stress upregulates the expression of stress-responsive genes in transgenic Arabidopsis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the molecular mechanism underlying the drought tolerance of \u003cem\u003eSlLEA_2-26\u003c/em\u003e, the relative expression levels of \u003cem\u003eSlLEA_2-26\u003c/em\u003e and the stress-responding genes \u003cem\u003eAtP5CS1\u003c/em\u003e, \u003cem\u003eAtCSD1\u003c/em\u003e, \u003cem\u003eAtNCED3\u003c/em\u003e, \u003cem\u003eAtRD26\u003c/em\u003e, and \u003cem\u003eAtRD29A\u003c/em\u003e were detected in WT and OE plants under drought stress. As expected, the mRAN accumulation of \u003cem\u003eSlLEA_2-26\u003c/em\u003e in OEs continuously increased under drought condition (Fig. 5a). The expression patterns of above five stress-responsive genes in OEs resembled that of \u003cem\u003eSlLEA_2-26\u003c/em\u003e, peaking at 14 days under drought stress with the extremely significant difference compared to the WT plants (Fig. 5b-f). While \u003cem\u003eAtP5CS1\u003c/em\u003e expression in transgenic plants reached approximately twice the WT level at drought 14\u003csup\u003eth\u003c/sup\u003e day, the expression of \u003cem\u003eAtCSD1\u003c/em\u003e in lines OE6, OE9, and OE11 was 1.04, 1.7, and 0.68 times higher than WT, respectively (Fig. 5b,c). Their expression levels slightly decreased after 3 days of rewatering. These results indicated that heterologous expression of \u003cem\u003eSlLEA_2-26\u003c/em\u003e can induce the upregulation of stress-associated genes in Arabidopsis under drought stress.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.6. Overexpression of SlLEA_2-26 enhances salt stress tolerance in transgenic Arabidopsis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo further investigate the effect of \u003cem\u003eSlLEA_2-26\u003c/em\u003e overexpression on stress tolerance to salt, WT and OE Arabidopsis plants were subjected to irrigation with 225 mM NaCl for 15 days.No significant differences were observed in growth phenotype, soluble sugar and proline content, ROS content, and antioxidant enzyme activities between OE and WT plants under normal growth condition (Fig. 6a-j). However, the WT plants showed more severe wilting than OEs under salt stress, the majority of leaves in the OEs remained green. (Fig. 6a). Throughout the entire salt stress process, the contents of soluble sugar and proline continuously increased, and both remained significantly higher level in OEs than that in the WT plants (Fig. 6b,c). At day 5 of stress, the soluble sugar content in the overexpression lines increased markedly to 17 mg·g\u003csup\u003e-1\u003c/sup\u003e FW (Fig. 6b). As NaCl stress treatment time extension, the contents of MDA, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, and O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e·-\u003c/sup\u003e gradually increased, while those in the OE lines consistently exhibited significantly lower levels than that in the WT (Fig. 6h-j). At 15\u003csup\u003eth\u003c/sup\u003e day of salt stress, the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e content in the OEs was approximately 80% that of the WT (Fig. 6i). Meanwhile, the O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e·-\u003c/sup\u003e content in lines OE6, OE9, and OE11 was reduced by 23.03%, 25%, and 31.65%, respectively, compared to the WT (Fig. 6j). Similarly, the activities of SOD, POD, APX, and CAT rapidly increased after salt stress, and higher activity levels of antioxidant enzymes were presented in OEs plants than those in the WT (Fig. 6d-g). Remarkably, the OE lines exhibited higher POD and CAT activities, with their activities rising sharply compared to the WT after stress exposure. Moreover, this difference was most significant at 15 days after salt stress, with POD activity elevated by over 50% and CAT activity increased nearly two folds, respectively (Fig. 6e,g).\u003c/p\u003e\n\u003cp\u003eThe results above indicated that \u003cem\u003eSlLEA_2-26\u003c/em\u003e-overexpression in Arabidopsis enhances salt tolerance by promoting the accumulation of osmotic regulatory substances, increasing the antioxidant enzyme activities, and alleviating oxidative damage caused by NaCl stress.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.7. Salt stress upregulates the expression of stress-responsive genes in transgenic Arabidopsis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe relative expression levels of \u003cem\u003eSlLEA_2-26\u003c/em\u003e and stress-responding genes were further detected in OE lines and WT plants under NaCl treatment to reveal the molecular mechanism of salt stress tolerance. As expected, salt stress significantly induced the upregulation of \u003cem\u003eSlLEA_2-26\u003c/em\u003e gene in OE Arabidopsis plants with the extension of stress treatment (Fig. 7a). In addition, the mRNA levels of five stress- associated genes including \u003cem\u003eAtP5CS1\u003c/em\u003e, \u003cem\u003eAtCSD1\u003c/em\u003e, \u003cem\u003eAtNCED3\u003c/em\u003e, \u003cem\u003eAtRD26\u003c/em\u003e, and \u003cem\u003eAtRD29A\u003c/em\u003e in Arabidopsis continuously increased with the extension of stress duration (Fig. 7b-f). Meanwhile, the OE plants exhibited significant higher expression level of stress-responding genes than that in WT. After 15 days of salt stress, transgenic plants showed a 59-89% increase in \u003cem\u003eAtP5CS1\u003c/em\u003e expression relative to WT, while \u003cem\u003eAtCSD1\u003c/em\u003e expression reached about 2.5 times the WT level (Fig. 7b,c). In conclusion, overexpression of \u003cem\u003eSlLEA_2-26\u003c/em\u003e in Arabidopsis can induce the upregulation of stress-responsive genes under salt stress.\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn this study, \u003cem\u003eSlLEA_2-26\u003c/em\u003e gene exhibited distinct tissue-specific expression patterns, with significantly higher transcript levels detected in flowers and fruits compared to other tissues (Fig. 2a). These observations align with prior reports indicating that \u003cem\u003eIpomoea pescaprae\u003c/em\u003e \u003cem\u003eIpLEA\u003c/em\u003e (Zheng JX et al. 2019) and rice \u003cem\u003eOsEm1\u003c/em\u003e (Yu J et al. 2016) display elevated transcriptional level in flowers and developing seeds, consistent with the conserved functional characteristics of \u003cem\u003eLEA\u003c/em\u003e genes. \u003cem\u003eSlLEA_2-26\u003c/em\u003e gene demonstrated differential responsiveness to diverse abiotic stresses, exhibiting marked upregulation under drought and salt stress conditions. Under heat and cold stress, transcript level reached up to six-fold higher than those under normal growth conditions, whereas its responsiveness to ABA was comparatively weak (Fig. 2b). The observed stress response patterns of \u003cem\u003eSlLEA_2-26\u003c/em\u003e gene to abiotic stresses are consistent with previous studies (Park SC et al. 2011; Zheng JX et al. 2019). Collectively, these results indicated that \u003cem\u003eSlLEA_2-26\u003c/em\u003e play a positive role under multiple stress conditions, thereby prompting further functional validation in transgenic Arabidopsis lines.\u003c/p\u003e\n\u003cp\u003eDrought and salt stress can significantly impair seed germination efficiency and inhibit seedling root elongation, both of which serve as critical physiological indicators for evaluating plant stress tolerance (Henry A 2013). Consistent with these findings, heterologous expression of cotton \u003cem\u003eGhLEA_2\u003c/em\u003e in Arabidopsis and overexpression of \u003cem\u003eSalvia miltiorrhiza\u003c/em\u003e \u003cem\u003eSmLEA2\u003c/em\u003e were demonstrated to significantly promote root growth in transgenic plants (Magwanga RO et al. 2018; Wang HQ et al. 2017). Similarly, overexpression of rice \u003cem\u003eOsEm1\u003c/em\u003e has been shown to enhance drought and salt tolerance in rice seedlings (Yu J et al. 2016). In this study, under drought and salt stress conditions, transgenic Arabidopsis lines overexpressing \u003cem\u003eSlLEA_2-26\u003c/em\u003e exhibited significantly higher seed germination rates, increased root lengths, and greater fresh weights compared to the WT plants (Fig. 3a-e). Furthermore, phenotypic analysis revealed pronounced morphological differences between WT and OE plants, with the latter displaying enhanced tolerance during stress stages (Fig. 4a, Fig. 6a). These observations collectively indicate that \u003cem\u003eSlLEA_2-26\u003c/em\u003e overexpression confers improved drought and salt tolerance in Arabidopsis during both germination and seedling establishment phases.\u003c/p\u003e\n\u003cp\u003eDrought and salinity can induce osmotic stress in plants, prompting cellular accumulation of osmoregulatory substances including soluble sugars and proline to mitigate adverse effects (Bao YQ et al. 2025). Proline serves a multifaceted role in stress adaptation: it can not only maintain the osmotic balance between cells and the environment but also act as a signaling molecule to activate various responses related to abiotic stress (Kavi Kishor PB and Sreenivasulu N 2014; Natarajan SK et al. 2012). In this study, under both drought and salt stress, transgenic Arabidopsis lines overexpressing \u003cem\u003eSlLEA_2-26\u003c/em\u003e exhibited substantial accumulation of soluble sugars and proline, with levels significantly exceeding those in WT plants (Fig. 4b,c; Fig. 6b,c). These findings indicate that \u003cem\u003eSlLEA_2-26\u003c/em\u003e overexpression enhances the synthesis of osmoregulatory compounds under drought and salt condition, thereby improving stress tolerance through osmotic adjustment. Both the proteins encoded by \u003cem\u003efoxtail millet\u003c/em\u003e \u003cem\u003eSiLEA14\u003c/em\u003e and tomato \u003cem\u003eSlLEA_2-26\u003c/em\u003e belong to atypical LEA proteins. Under drought or salt stress, proline and soluble sugar accumulation patterns in \u003cem\u003eSiLEA14\u003c/em\u003e- overexpressing \u003cem\u003efoxtail mille\u003c/em\u003et were consistent with those observed in this study, demonstrating conserved regulatory mechanisms. These proteins maintain cellular water balance by enhancing osmotic adjustment capacity and alleviating drought-induced cellular damage (Wang MZ et al. 2014), corroborating findings from studies on \u003cem\u003eLEA\u003c/em\u003e genes overexpression in alfalfa (Jia HL et al. 2020; Ma WX et al. 2025), ginseng (Wang Q et al. 2024), and muskmelon (Aduse Poku S et al. 2020). Notably, proline expression levels reached exceptionally high values under salt stress, aligning with transcriptional analysis data (Fig. 7b). This observation further substantiates that \u003cem\u003eSlLEA_2-26\u003c/em\u003e exhibits a robust response to salt stress, highlighting its potential as a key regulator in salinity adaptation.\u003c/p\u003e\n\u003cp\u003eDrought and salt stresses induce excessive accumulation of ROS in plants, which can trigger biomembrane system damage through lipid peroxidation, impairing photosynthesis, disrupting respiratory metabolism, and ultimately leading to plant mortality (Basu S et al. 2021). The levels of O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e·-\u003c/sup\u003e and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e serve as direct indicators of ROS accumulation, whereas MDA content reflects the extent of membrane lipid peroxidation (Hasanuzzaman M and Fujita M 2022). During the long evolutionary process, Plants have evolved an antioxidant defense to scavenge ROS and mitigate oxidative damage, thereby maintaining physiological homeostasis. Under stress condition, antioxidant enzymes including SOD, APX, POD, and CAT are activated to counteract abiotic stresses (Sofo A et al. 2015). SOD catalyzes the dismutation of O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e·-\u003c/sup\u003e into H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and O\u003csub\u003e2\u003c/sub\u003e, while APX, POD, and CAT collectively scavenge H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, thereby reducing ROS level and alleviating biomembrane damage.\u003c/p\u003e\n\u003cp\u003eIn this study, under both drought and salt stress condition, the contents of MDA, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, and O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e·-\u003c/sup\u003e in transgenic Arabidopsis lines were significantly reduced compared to WT plants, with the decline becoming more pronounced as the stress duration increased (Fig. 4h-j; Fig. 6h-j). This suggests diminished oxidative damage in transgenic plants. Concurrently, the activities of antioxidant enzymes including SOD, POD, APX, and CAT were significantly higher in OE Arabidopsis lines than those in WT plants (Fig. 4d-g, Fig. 6d-g). These findings align with previous reports, where heterologous overexpression of the muskmelon \u003cem\u003eCmLEA-S\u003c/em\u003e gene in tobacco (Aduse Poku S et al. 2020) and the peanut \u003cem\u003eAhLEA2\u003c/em\u003e gene in Arabidopsis (Li C et al. 2022) similarly enhanced antioxidant enzyme activity and reduced MDA accumulation, accompanied by improved drought and salt stress tolerance in transgenic plants. Collectively, the data indicate that \u003cem\u003eSlLEA_2-26\u003c/em\u003e overexpression enhances antioxidant enzyme activity, thereby improving ROS scavenging capacity and effectively mitigating oxidative damage in transgenic Arabidopsis under drought and salt stress.\u003c/p\u003e\n\u003cp\u003eAbiotic stress triggers the transcriptional activation of numerous stress-responsive genes. Zheng et al (2019) reported that heterologous overexpression of \u003cem\u003eIpomoea pescaprae\u003c/em\u003e \u003cem\u003eIpLEA\u003c/em\u003e in Arabidopsis induced the expression of stress-associated genes including \u003cem\u003eCSD1\u003c/em\u003e, \u003cem\u003eNCED3\u003c/em\u003e, and \u003cem\u003eRD29A\u003c/em\u003e, consequently enhancing abiotic stress tolerance in transgenic plants. To elucidate the molecular mechanisms underlying \u003cem\u003eSlLEA_2-26\u003c/em\u003e-mediated drought and salt stress responses, this study quantified the expression levels of five stress tolerance-related genes. The synthetase encoded by \u003cem\u003eP5CS\u0026nbsp;\u003c/em\u003eis the rate-limiting enzyme in the biosynthesis of proline (Amini S et al. 2015). \u003cem\u003eCSD1\u003c/em\u003e encodes SOD which catalyzes the dismutation of O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e·-\u003c/sup\u003e into H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and O\u003csub\u003e2\u003c/sub\u003e (Cui LJ et al. 2015). \u003cem\u003eNCED3\u003c/em\u003e encodes a key enzyme in the biosynthesis of ABA, a pivotal phytohormone orchestrating abiotic stress responses such as drought (Nakashima K and Yamaguchi-Shinozaki K 2013). \u003cem\u003eRD26\u003c/em\u003e encodes an NAC transcription factor integral to ABA-mediated dehydration signaling pathways (Fujita M et al. 2004). \u003cem\u003eRD29A\u003c/em\u003e responds to multiple abiotic stresses including drought, salinity, high temperature, and ABA, encoding a highly hydrophilic protein that protects cells from dehydration damage (Yamaguchi-Shinozaki K et al. 1992). In this study, all five stress-responsive genes exhibited significantly elevated expression levels in \u003cem\u003eSlLEA_2-26\u003c/em\u003e-overexpressing Arabidopsis compared to WT plants (Fig. 5b-f, Fig. 7b-f). Notably, the upregulation of \u003cem\u003eAtP5CS1\u003c/em\u003e and \u003cem\u003eAtCSD1\u003c/em\u003e correlated with increased proline accumulation and SOD activity under drought and salt stress (Fig. 4c-d, Fig. 5b-c, Fig. 6c-d, Fig. 7b-c). These findings indicate that overexpression of \u003cem\u003eSlLEA_2-26\u003c/em\u003e can induce the transcriptional activation of stress-responsive genes, thereby augmenting antioxidant enzyme activity and enhancing ROS scavenging efficiency. Concurrently, it activates ABA signaling pathway components, modulating Arabidopsis adaptive responses to environmental stress. Consistent with these observations, studies on tobacco overexpressing the peanut \u003cem\u003eAdLEA\u003c/em\u003e demonstrated that upregulation of key stress response pathway genes confers enhanced abiotic stress tolerance in transgenic plants (Sharma A et al. 2016). This cross-species conservation underscores the pivotal role of LEA proteins in orchestrating plant stress adaptation.\u003c/p\u003e\n\u003cp\u003eIn conclusion, the overexpression of the \u003cem\u003eSlLEA_2-26\u003c/em\u003e gene in Arabidopsis triggers the upregulation stress-responsive genes under drought and salt stress, thereby mediating plant stress tolerance through dual regulatory pathways. Firstly, it promotes the biosynthesis of osmolytes such as proline via osmoprotectant synthesis, thereby maintaining cellular osmotic equilibrium and water homeostasis. Secondly, it synergistically enhances the activity of the antioxidant enzyme system, mitigating ROS-induced membrane damage and alleviating stress-related cellular dysfunction. Collectively, these mechanisms confer improved abiotic stress tolerance in transgenic plants. This study characterized the functional role of the \u003cem\u003eSlLEA_2-26\u003c/em\u003e gene by analyzing stress responses in transgenic Arabidopsis, and demonstrated its positive contribution to abiotic stress adaptation. Future research will focus on dissecting the regulatory networks of \u003cem\u003eSlLEA_2-26\u003c/em\u003e in tomato to elucidate its species-specific functions during abiotic stress.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eROS, Reactive oxygen species; LEA, Late embryogenesis abundant; qRT-PCR, Quantitative real-time PCR; TBA, Thiobarbituric acid; NBT, Nitroblue tetrazolium; OE, Overexpression; WT, Wild type; MDA, Malondialdehyde; H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, Hydrogen peroxide; O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e.-\u003c/sup\u003e; Superoxide anion; CAT, Catalase; POD, peroxidase; SOD, Superoxide dismutase; APX, Ascorbate peroxidase; ANOVA, Analysis of variance; DHN,Dehydrin; SMP, Seed maturation protein; NJ, Neighbor-Joining\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDeclaration of c\u003c/strong\u003e\u003cstrong\u003eompeting interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest related to the content of this article.\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Science and Technology Innovation Enhancement Project of Shanxi Agricultural University (CXGC2025066).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAppendix A. Supplementary data\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupplementary data to this article can be found online at https://--------\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization:\u0026nbsp;W.W., Z.Y., X.Z.; Investigation and writing–original draft preparation: Z.Y., Y.L.; Validation: X.Z., S.W.; Visualization: Y.Y., Y.L.; Formal analysis: W.W., Y.Y.; Resources: D.Y., L.L.; Writing–review and editing: W.W., Z.Y.; Supervision and funding acquisition: W.W.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHenry A (2013) IRRI\u0026rsquo;s drought stress research in rice with emphasis on roots: accomplishments over the last 50 years. Plant Root 7:92\u0026ndash;106. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3117/plantroot.7.92\u003c/span\u003e\u003cspan address=\"10.3117/plantroot.7.92\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAduse Poku S, Nkachukwu Chukwurah P, Aung HH, Nakamura I (2020) Over-Expression of a Melon Y3SK2-Type \u003cem\u003eLEA\u003c/em\u003e Gene Confers Drought and Salt Tolerance in Transgenic Tobacco Plants. Plants (Basel) 9:1749. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/plants9121749\u003c/span\u003e\u003cspan address=\"10.3390/plants9121749\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAli MY, Sina AA, Khandker SS, Neesa L, Tanvir EM, Kabir A, Khalil MI, Gan SH (2020) Nutritional Composition and Bioactive Compounds in Tomatoes and Their Impact on Human Health and Disease: A Review. Foods (Basel Switzerland) 10:45. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/foods10010045\u003c/span\u003e\u003cspan address=\"10.3390/foods10010045\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAmara I, Capellades M, Ludevid MD, Pag\u0026egrave;s M, Goday A (2013) Enhanced water stress tolerance of transgenic maize plants over-expressing \u003cem\u003eLEA Rab28\u003c/em\u003e gene. J Plant Physiol 170:864\u0026ndash;873. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.jplph.2013.01.004\u003c/span\u003e\u003cspan address=\"10.1016/j.jplph.2013.01.004\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAmini S, Ghobadi C, Yamchi A (2015) Proline accumulation and osmotic stress: an overview of P5CS gene in plants. J Plant Mol Breed 3:44\u0026ndash;55. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.22058/jpmb.2015.17022\u003c/span\u003e\u003cspan address=\"10.22058/jpmb.2015.17022\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBanerjee A, Roychoudhury A (2016) Group II late embryogenesis abundant (LEA) proteins: structural and functional aspects in plant abiotic stress. Plant Growth Regul 79:1\u0026ndash;17. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s10725-015-0113-3\u003c/span\u003e\u003cspan address=\"10.1007/s10725-015-0113-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBao YQ, Zeng Y, Zhao Y, Liu Y, Zhao YQ, Xiao LH, Wu ZG, Zheng YH, Jin P (2025) Phytosulfokine α treatment induces osmotic regulation to alleviate postharvest chilling injury of peach fruit by PpbHLH62. Postharvest Biol Technol 230:113746. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.postharvbio.2025.113746\u003c/span\u003e\u003cspan address=\"10.1016/j.postharvbio.2025.113746\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBasu S, Kumari S, Kumar P, Kumar G, Rajwanshi R (2021) Redox imbalance impedes photosynthetic activity in rice by disrupting cellular membrane integrity and induces programmed cell death under submergence. Physiol Plant 172:1764\u0026ndash;1778. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/ppl.13387\u003c/span\u003e\u003cspan address=\"10.1111/ppl.13387\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBattaglia M, Olvera-Carrillo Y, Garciarrubio A, Campos F, Covarrubias AA (2008) The enigmatic LEA proteins and other hydrophilins. Plant Physiol 148:6\u0026ndash;24. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1104/pp.108.120725\u003c/span\u003e\u003cspan address=\"10.1104/pp.108.120725\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBindschedler LV, Minibayeva F, Gardner SL, Gerrish C, Davies DR, Bolwell GP (2001) Early signalling events in the apoplastic oxidative burst in suspension cultured French bean cells involve cAMP and Ca\u003csup\u003e2+\u003c/sup\u003e. New Phytol 151:185\u0026ndash;194. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1046/j.1469-8137.2001.00170.x\u003c/span\u003e\u003cspan address=\"10.1046/j.1469-8137.2001.00170.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBurton-Freeman B, Sesso HD (2014) Whole food versus supplement: comparing the clinical evidence of tomato intake and lycopene supplementation on cardiovascular risk factors. Adv Nutr 5:457\u0026ndash;485. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3945/an.114.005231\u003c/span\u003e\u003cspan address=\"10.3945/an.114.005231\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCelik Altunoglu Y, Baloglu P, Yer EN, Pekol S, Baloglu MC (2016) Identification and expression analysis of \u003cem\u003eLEA\u003c/em\u003e gene family members in cucumber genome. Plant Growth Regul 80:225\u0026ndash;241. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s10725-016-0160-4\u003c/span\u003e\u003cspan address=\"10.1007/s10725-016-0160-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChakrabortee S, Boschetti C, Walton LJ, Sarkar S, Rubinsztein DC, Tunnacliffe A (2007) Hydrophilic protein associated with desiccation tolerance exhibits broad protein stabilization function. Proc Natl Acad Sci USA 104:18073\u0026ndash;18078. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1073/pnas.0706964104\u003c/span\u003e\u003cspan address=\"10.1073/pnas.0706964104\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen TZ, Zhang BL (2016) Measurements of Proline and Malondialdehyde Content and Antioxidant Enzyme Activities in Leaves of Drought Stressed Cotton. Bio-protocol 6:e1913. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.21769/bioprotoc.1913\u003c/span\u003e\u003cspan address=\"10.21769/bioprotoc.1913\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eClough SJ, Bent AF (1998) Floral dip: a simplified method for \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated transformation of \u003cem\u003eArabidopsis thaliana\u003c/em\u003e. Plant J 16:735\u0026ndash;743. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1046/j.1365-313x.1998.00343.x\u003c/span\u003e\u003cspan address=\"10.1046/j.1365-313x.1998.00343.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCui LJ, Huang Q, Yan B, Wang Y, Qian ZY, Pan JX, Kai GY (2015) Molecular cloning and expression analysis of a Cu/Zn SOD gene (\u003cem\u003eBcCSD1\u003c/em\u003e) from \u003cem\u003eBrassica campestris\u003c/em\u003e ssp. \u003cem\u003echinensis\u003c/em\u003e. Food chemistry 186:306\u0026ndash;311. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.foodchem.2014.07.121\u003c/span\u003e\u003cspan address=\"10.1016/j.foodchem.2014.07.121\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDure L 3rd, Greenway SC, Galau GA (1981) Developmental biochemistry of cottonseed embryogenesis and germination: changing messenger ribonucleic acid populations as shown by in vitro and in vivo protein synthesis. Biochemistry 20:4162\u0026ndash;4168. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1021/bi00517a033\u003c/span\u003e\u003cspan address=\"10.1021/bi00517a033\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFerdous J, Hussain SS, Shi BJ (2015) Role of microRNAs in plant drought tolerance. Plant Biotechnol J 13:293\u0026ndash;305. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/pbi.12318\u003c/span\u003e\u003cspan address=\"10.1111/pbi.12318\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFujita M, Fujita Y, Maruyama K, Seki M, Hiratsu K, Ohme-Takagi M, Tran LS, Yamaguchi‐Shinozaki K, Shinozaki K (2004) A dehydration‐induced NAC protein, RD26, is involved in a novel ABA‐dependent stress‐signaling pathway. Plant J 39:863\u0026ndash;876. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/j.1365-313X.2004.02171.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1365-313X.2004.02171.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGalau GA, Wang HY, Hughes DW (1993) Cotton \u003cem\u003eLea5\u003c/em\u003e and \u003cem\u003eLea14\u003c/em\u003e encode atypical late embryogenesis-abundant proteins. Plant Physiol 101:695\u0026ndash;696. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1104/pp.101.2.695\u003c/span\u003e\u003cspan address=\"10.1104/pp.101.2.695\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGrelet J, Benamar A, Teyssier E, Avelange-Macherel MH, Grunwald D, Macherel D (2005) Identification in pea seed mitochondria of a late-embryogenesis abundant protein able to protect enzymes from drying. Plant Physiol 137:157\u0026ndash;167. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1104/pp.104.052480\u003c/span\u003e\u003cspan address=\"10.1104/pp.104.052480\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGrivna ST, Beyret E, Wang Z, Lin HF (2006) A novel class of small RNAs in mouse spermatogenic cells. Genes Dev 20:1709\u0026ndash;1714. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1101/gad.1434406\u003c/span\u003e\u003cspan address=\"10.1101/gad.1434406\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHasanuzzaman M, Fujita M (2022) Plant Oxidative Stress: Biology, Physiology and Mitigation. Plants (Basel) 11:1185. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/plants11091185\u003c/span\u003e\u003cspan address=\"10.3390/plants11091185\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHundertmark M, Hincha DK (2008) LEA (late embryogenesis abundant) proteins and their encoding genes in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e. BMC Genomics 9:118. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/1471-2164-9-118\u003c/span\u003e\u003cspan address=\"10.1186/1471-2164-9-118\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIM Petyaev (2016) Lycopene Deficiency in Ageing and Cardiovascular Disease. Oxid Med Cell Longev 2016:6. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1155/2016/3218605\u003c/span\u003e\u003cspan address=\"10.1155/2016/3218605\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJia HL, Wang XM, Shi YH, Wu XM, Wang YQ, Liu JN, Fang ZH, Li CY, Dong KH (2020) Overexpression of \u003cem\u003eMedicago sativa LEA4-4\u003c/em\u003e can improve the salt, drought, and oxidation resistance of transgenic \u003cem\u003eArabidopsis\u003c/em\u003e. PLoS ONE 15:e0234085. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1371/journal.pone.0234085\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0234085\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJisha KC, Vijayakumari K, Puthur JT (2013) Seed priming for abiotic stress tolerance: an overview. Acta Physiol Plant 35:1381\u0026ndash;1396. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s11738-012-1186-5\u003c/span\u003e\u003cspan address=\"10.1007/s11738-012-1186-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKaur N, Sharma I, Kirat K, Pati PK (2016) Detection of Reactive Oxygen Species in \u003cem\u003eOryza sativa\u003c/em\u003e L.(rice). Bio-protocol 6:e2061-e2061. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.21769/BioProtoc.2061\u003c/span\u003e\u003cspan address=\"10.21769/BioProtoc.2061\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKavi Kishor PB, Sreenivasulu N (2014) Is proline accumulation per se correlated with stress tolerance or is proline homeostasis a more critical issue? Plant Cell Environ 37:300\u0026ndash;311. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/pce.12157\u003c/span\u003e\u003cspan address=\"10.1111/pce.12157\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim HS, Lee JH, Kim JJ, Kim CH, Jun SS, Hong YN (2005) Molecular and functional characterization of \u003cem\u003eCaLEA6\u003c/em\u003e, the gene for a hydrophobic LEA protein from \u003cem\u003eCapsicum annuum\u003c/em\u003e. Gene 344:115\u0026ndash;123. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.gene.2004.09.012\u003c/span\u003e\u003cspan address=\"10.1016/j.gene.2004.09.012\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKovacs D, Kalmar E, Torok Z, Tompa P (2008) Chaperone activity of ERD10 and ERD14, two disordered stress-related plant proteins. Plant Physiol 147:381\u0026ndash;390. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1104/pp.108.118208\u003c/span\u003e\u003cspan address=\"10.1104/pp.108.118208\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi C, Yan C, Sun Q, Wang J, Yuan C, Mou Y, Shan S, Zhao X (2022) Proteomic profiling of \u003cem\u003eArachis hypogaea\u003c/em\u003e in response to drought stress and overexpression of \u003cem\u003eAhLEA2\u003c/em\u003e improves drought tolerance. Plant Biol 24:75\u0026ndash;84. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/plb.13351\u003c/span\u003e\u003cspan address=\"10.1111/plb.13351\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi SF, Meng HJ, Yang YF, Zhao JN, Xia YX, Wang SL, Wang F, Zheng GS, Li JB (2025) Overexpression of \u003cem\u003eAtruLEA1\u003c/em\u003e from \u003cem\u003eAcer truncatum\u003c/em\u003e Bunge Enhanced \u003cem\u003eArabidopsis\u003c/em\u003e Drought and Salt Tolerance by Improving ROS-Scavenging Capability. Plants (Basel) 14:117. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/plants14010117\u003c/span\u003e\u003cspan address=\"10.3390/plants14010117\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu Y, Wang L, Xing X, Sun LP, Pan JW, Kong XP, Zhang MY, Li DQ (2013) ZmLEA3, a multifunctional group 3 LEA protein from maize (\u003cem\u003eZea mays\u003c/em\u003e L.), is involved in biotic and abiotic stresses. Plant Cell Physiol 54:944\u0026ndash;959. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/pcp/pct047\u003c/span\u003e\u003cspan address=\"10.1093/pcp/pct047\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMa WX, Wen X, Zhao L, Dong SW, Luo D, Zhou Q, Fang LF, Liu WX, Liu ZP (2025) \u003cem\u003eMsLEA_3\u0026ndash;6\u003c/em\u003e overexpression enhances drought tolerance in \u003cem\u003eMedicago sativa\u003c/em\u003e L. BMC Plant Biol 25:1482. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s12870-025-07101-9\u003c/span\u003e\u003cspan address=\"10.1186/s12870-025-07101-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMagwanga RO, Lu P, Kirungu JN, Dong Q, Hu YG, Zhou ZL, Cai XY, Wang XX, Hou YQ, Wang KB (2018) Cotton Late Embryogenesis Abundant (\u003cem\u003eLEA2\u003c/em\u003e) Genes Promote Root Growth and Confer Drought Stress Tolerance in Transgenic \u003cem\u003eArabidopsis thaliana\u003c/em\u003e. G3(Bethesda):2781\u0026ndash;2803. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1534/g3.118.200423\u003c/span\u003e\u003cspan address=\"10.1534/g3.118.200423\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMagwanga RO, Lu P, Kirungu JN, Lu HJ, Wang XX, Cai XY, Zhou ZL, Zhang ZM, Salih H, Wang KB, Liu F (2018) Characterization of the late embryogenesis abundant (LEA) proteins family and their role in drought stress tolerance in upland cotton. BMC Genet 19:6. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s12863-017-0596-1\u003c/span\u003e\u003cspan address=\"10.1186/s12863-017-0596-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNakashima K, Yamaguchi-Shinozaki K (2013) ABA signaling in stress-response and seed development. Plant Cell Rep 32:959\u0026ndash;970. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s00299-013-1418-1\u003c/span\u003e\u003cspan address=\"10.1007/s00299-013-1418-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNatarajan SK, Zhu WD, Liang XW, Zhang L, Demers AJ, Zimmerman MC, Simpson MA, Becker DF (2012) Proline dehydrogenase is essential for proline protection against hydrogen peroxide-induced cell death. Free Radic Biol Med 53:1181\u0026ndash;1191. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.freeradbiomed.2012.07.002\u003c/span\u003e\u003cspan address=\"10.1016/j.freeradbiomed.2012.07.002\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePark SC, Kim YH, Jeong JC, Kim CY, Lee HS, Bang JW, Kwak SS (2011) Sweetpotato late embryogenesis abundant 14 (\u003cem\u003eIbLEA14\u003c/em\u003e) gene influences lignification and increases osmotic-and salt stress-tolerance of transgenic calli. Planta 233:621\u0026ndash;634. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s00425-010-1326-3\u003c/span\u003e\u003cspan address=\"10.1007/s00425-010-1326-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePerveen R, Suleria HAR, Anjum FM, Butt MS, Pasha I, Ahmad S (2015) Tomato (\u003cem\u003eSolanum lycopersicum\u003c/em\u003e) Carotenoids and Lycopenes Chemistry; Metabolism, Absorption, Nutrition, and Allied Health Claims\u0026mdash;A Comprehensive Review. Crit Rev Food Sci Nutr 55:919\u0026ndash;929. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1080/10408398.2012.657809\u003c/span\u003e\u003cspan address=\"10.1080/10408398.2012.657809\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eReyes JL, Campos F, Wei H, Arora R, Yang Y, Karlson DT, Covarrubias AA (2008) Functional dissection of hydrophilins during in \u003cem\u003evitro\u003c/em\u003e freeze protection. Plant Cell Environ 31:1781\u0026ndash;1790. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/j.1365-3040.2008.01879.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1365-3040.2008.01879.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRodr\u0026iacute;guez-Valent\u0026iacute;n R, Battaglia M, Campos F, Sol\u0026oacute;rzano RM, Rosales MA, Covarrubias AA (2014) Group 6 Late Embryogenesis Abundant (LEA) Proteins in Monocotyledonous Plants: Genomic Organization and Transcript Accumulation Patterns in Response to Stress in \u003cem\u003eOryza sativa\u003c/em\u003e. Plant Mol Biology Report 32:198\u0026ndash;208. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s11105-013-0641-9\u003c/span\u003e\u003cspan address=\"10.1007/s11105-013-0641-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSharma A, Kumar D, Kumar S, Rampuria S, Reddy AR, Kirti PB (2016) Ectopic Expression of an Atypical Hydrophobic Group 5 LEA Protein from Wild Peanut, \u003cem\u003eArachis diogoi\u003c/em\u003e Confers Abiotic Stress Tolerance in Tobacco. PLoS ONE 11:e0150609. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1371/journal.pone.0150609\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0150609\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSofo A, Scopa A, Nuzzaci M, Vitti A (2015) Ascorbate Peroxidase and Catalase Activities and Their Genetic Regulation in Plants Subjected to Drought and Salinity Stresses. Int J Mol Sci 16:13561\u0026ndash;13578. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/ijms160613561\u003c/span\u003e\u003cspan address=\"10.3390/ijms160613561\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTolleter D, Jaquinod M, Mangavel C, Passirani C, Saulnier P, Manon S, eyssier E, Payet N, Avelange-Macherel MH, Macherel D (2007) Structure and function of a mitochondrial late embryogenesis abundant protein are revealed by desiccation. Plant Cell 19:1580\u0026ndash;1589. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1105/tpc.107.050104\u003c/span\u003e\u003cspan address=\"10.1105/tpc.107.050104\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWaadt R, Seller CA, Hsu PK, Takahashi Y, Munemasa S, Schroeder JI (2022) Plant hormone regulation of abiotic stress responses. Nat Rev Mol Cell Biol 23:680\u0026ndash;694. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41580-022-00479-6\u003c/span\u003e\u003cspan address=\"10.1038/s41580-022-00479-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang HQ, Wu YC, Yang XB, Guo XR, Cao XY (2017) \u003cem\u003eSmLEA2\u003c/em\u003e, a gene for late embryogenesis abundant protein isolated from \u003cem\u003eSalvia miltiorrhiza\u003c/em\u003e, confers tolerance to drought and salt stress in \u003cem\u003eEscherichia coli\u003c/em\u003e and \u003cem\u003eS. miltiorrhiza\u003c/em\u003e. Protoplasma 254:685\u0026ndash;696. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s00709-016-0981-z\u003c/span\u003e\u003cspan address=\"10.1007/s00709-016-0981-z\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang MZ, Li P, Li C, Pan YL, Jiang XY, Zhu DY, Zhao Q, Yu JJ (2014) SiLEA14, a novel atypical LEA protein, confers abiotic stress resistance in foxtail millet. BMC Plant Biol 14:290. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s12870-014-0290-7\u003c/span\u003e\u003cspan address=\"10.1186/s12870-014-0290-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Q, Lei XJ, Wang YH, Di P, Meng XR, Peng WY, Rong JB, Wang YP (2024) Genome-wide identification of the \u003cem\u003eLEA\u003c/em\u003e gene family in \u003cem\u003ePanax ginseng\u003c/em\u003e: Evidence for the role of \u003cem\u003ePgLEA2-50\u003c/em\u003e in plant abiotic stress response. Plant Physiol Biochem 212:108742. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.plaphy.2024.108742\u003c/span\u003e\u003cspan address=\"10.1016/j.plaphy.2024.108742\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang WB, Kim YH, Lee HS, Kim KY, Deng XP, Kwak SS (2009) Analysis of antioxidant enzyme activity during germination of alfalfa under salt and drought stresses. Plant Physiol Biochem 47:570\u0026ndash;577. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.plaphy.2009.02.009\u003c/span\u003e\u003cspan address=\"10.1016/j.plaphy.2009.02.009\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYamaguchi-Shinozaki K, Urao S, Koizumi M, Shinozaki K (1992) Molecular Cloning and Characterization of 9 cDNAs for Genes That Are Responsive to Desiccation in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e: SequenceAnalysis of One cDNA Clone That Encodes a Putative Transmembrane Channel Protein. Plant Cell Physiol 33:217\u0026ndash;224. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/oxfordjournals.pcp.a078243\u003c/span\u003e\u003cspan address=\"10.1093/oxfordjournals.pcp.a078243\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu J, Lai YM, Wu X, Wu G, Guo CK (2016) Overexpression of \u003cem\u003eOsEm1\u003c/em\u003e encoding a group I LEA protein confers enhanced drought tolerance in rice. Biochem Biophys Res Commun 478:703\u0026ndash;709. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.bbrc.2016.08.010\u003c/span\u003e\u003cspan address=\"10.1016/j.bbrc.2016.08.010\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZaman Khan N, Lal S, Ali W, Aasim M, Mumtaz S, Kamil A, Shad Bibi N (2020) Distribution and Classification of Dehydrins in Selected Plant Species Using Bioinformatics Approach. Iran J Biotechnol 18:e2680. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.30498/IJB.2020.2680\u003c/span\u003e\u003cspan address=\"10.30498/IJB.2020.2680\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZheng JX, Su HX, Lin RY, Zhang H, Xia KF, Jian SG, Zhang M (2019) Isolation and characterization of an atypical \u003cem\u003eLEA\u003c/em\u003e gene (\u003cem\u003eIpLEA\u003c/em\u003e) from \u003cem\u003eIpomoea pes-caprae\u003c/em\u003e conferring salt/drought and oxidative stress tolerance. Sci Rep 9:14838. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41598-019-50813-w\u003c/span\u003e\u003cspan address=\"10.1038/s41598-019-50813-w\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-plant-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jpre","sideBox":"Learn more about [Journal of Plant Research](http://link.springer.com/journal/10265)","snPcode":"10265","submissionUrl":"https://www.editorialmanager.com/jpre/default2.aspx","title":"Journal of Plant Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Abiotic stress, Late embryogenesis abundant protein, SlLEA_2–26, Stress tolerance, Tomato","lastPublishedDoi":"10.21203/rs.3.rs-8801747/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8801747/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eLate embryogenesis abundant (LEA) proteins are pivotal in conferring cellular tolerance to abiotic stresses and sustaining plant growth and development. However, systematic functional characterization of the tomato \u003cem\u003eSlLEA_2\u003c/em\u003e gene family remains limited. To elucidate the role of tomato \u003cem\u003eSlLEA_2\u0026ndash;26\u003c/em\u003e in abiotic stress responses, this study cloned its full-length cDNA. Quantitative real-time PCR (qRT-PCR) analysis revealed that \u003cem\u003eSlLEA_2\u0026ndash;26\u003c/em\u003e exhibits predominant expression in flowers and fruits, and is strongly induced by drought, salt, Cu\u003csup\u003e2+\u003c/sup\u003e, and Pb\u003csup\u003e2+\u003c/sup\u003e stresses. Three homozygous Arabidopsis T\u003csub\u003e3\u003c/sub\u003e \u003cem\u003eSlLEA_2-26\u003c/em\u003e-overexpression lines were generated and confirmed via genomic PCR. Under drought and salt stress, T\u003csub\u003e3\u003c/sub\u003e Arabidopsis \u003cem\u003eSlLEA_2-26\u003c/em\u003e-overexpressing lines demonstrated significantly enhanced seed germination rates, root elongation, and fresh weights wild type (WT) plants, indicating improved stress tolerance during early seedling development. Furthermore, transgenic plants accumulated higher levels of soluble sugar and proline, and displayed elevated antioxidant enzyme activity compared to the WT, whereas contents of malondialdehyde (MDA) and reactive oxygen species (ROS) were markedly reduced relative to WT. qRT-PCR analysis confirmed the significant upregulation of \u003cem\u003eSlLEA_2\u0026ndash;26\u003c/em\u003e in transgenic lines under drought and salt stress conditions, accompanied by elevated expression of \u003cem\u003eAtP5CS1\u003c/em\u003e, \u003cem\u003eAtCSD1\u003c/em\u003e, \u003cem\u003eAtRD29A\u003c/em\u003e, \u003cem\u003eAtRD26\u003c/em\u003e, and \u003cem\u003eAtNCED3\u003c/em\u003e. Collectively, these results demonstrate that \u003cem\u003eSlLEA_2\u0026ndash;26\u003c/em\u003e overexpression enhances drought and salt stress tolerance in Arabidopsis by promoting the accumulation of osmoregulatory substances, augmenting antioxidant defense capacity, and activating stress-responsive genes expression. This study provides a theoretical foundation and valuable genetic resources for breeding stress-tolerant tomatoes and other crops.\u003c/p\u003e","manuscriptTitle":"Overexpression of the tomato SlLEA_2-26 gene enhances the tolerance to drought and salt stresses in Arabidopsis thaliana","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-16 09:59:03","doi":"10.21203/rs.3.rs-8801747/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2026-03-16T08:08:47+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2026-02-25T23:01:23+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-10T21:49:10+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-09T11:50:46+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Plant Research","date":"2026-02-05T21:08:52+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-plant-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jpre","sideBox":"Learn more about [Journal of Plant Research](http://link.springer.com/journal/10265)","snPcode":"10265","submissionUrl":"https://www.editorialmanager.com/jpre/default2.aspx","title":"Journal of Plant Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"2732f605-b85e-4c4e-af70-d1ea2d04563b","owner":[],"postedDate":"February 16th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-18T10:48:33+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-16 09:59:03","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8801747","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8801747","identity":"rs-8801747","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}