A SMALL AUXIN UP-REGULATED RNA Gene Isolated from Watermelon (ClSAUR1) Positively Modulates the Chilling Stress Response in Tobacco via Multiple Signaling Pathways

Abstract

SMALL AUXIN UP-REGULATED RNA (SAURs) genes are acknowledged as auxin-responsive genes that play crucial roles in modulating adaptive growth under abiotic stress conditions. Low temperatures constitute a primary limiting factor that significantly impairs the development, growth, and fruit quality of watermelon plants during the winter and spring seasons. Despite their potential importance, SAURs have not yet been thoroughly investigated or characterized in watermelon. In this study, we identified a positive regulator of the chilling stress response among watermelon SAURs, designated as ClSAUR1. Subcellular localization analysis demonstrated that the protein is directed to both the nucleus and cytoplasm. Quantitative real-time PCR (qRT-PCR) analysis indicated that ClSAUR1 is ubiquitously expressed across various watermelon tissues, with pronounced expression in the roots and leaves. Moreover, qRT-PCR and promoter::β-glucuronidase (GUS) staining assays revealed that the expression of ClSAUR1 is significantly upregulated in response to exogenous abscisic acid (ABA) and chilling stress. The overexpression of ClSAUR1 in tobacco lines was contrasted and analyzed, revealing an increased tolerance to chilling stress. This was evidenced by a reduced degree of wilting and chlorosis compared to wild-type (WT) plants. Furthermore, the overexpressed lines showed reduced reactive oxygen species (ROS) accumulation and increased antioxidant enzyme activity. The qRT-PCR results further indicated that the expression levels of genes associated with abscisic acid (ABA), antioxidant enzymes, and CBF–COR cold-responsive pathways were upregulated in the transgenic tobacco lines. This study provides new insights into the role of ClSAURs in enhancing the cold resistance of watermelon.

1. Introduction

Auxin plays a crucial role in the regulation of plant growth, development, and adaptation to changing environmental conditions. Notably, SMALL AUXIN UP-REGULATED RNA (SAURs) genes are primary auxin-responsive genes that are integral to the auxin signaling pathway and are rapidly induced following auxin treatment.

Previous studies have demonstrated that certain SAUR genes are involved in the transport of plant auxin, contribute to the growth and development of various organs, and play roles in the plant stress response [1]. At elevated temperatures, the SAUR19 gene operates downstream of PIF4 to modulate hypocotyl elongation [2]. Additionally, the overexpression of AtSAUR19 has been shown to induce drought hypersensitivity in tomato plants [3]. Arabidopsis AtSAUR55 positively modulates hydrogen ion excretion through plasma membrane H⁺-ATPase 2, thereby enhancing aluminum tolerance by facilitating malate secretion from the roots [4]. Arabidopsis AtSAUR41, AtSAUR32, and poplar PtSAUR8 enhance plant drought resistance via abscisic acid (ABA)-mediated pathways, whereas AtSAUR19, peanut AhSAUR3, and AnSAUR50 negatively regulate drought resistance in plants [3,5,6,7,8,9,10]. The overexpression of wheat TaSAUR78 has been shown to improve tolerance to salt, drought, and freezing stresses in transgenic Arabidopsis [11]. Nonetheless, there is a paucity of research concerning the role of SAURs in the response to chilling stress.

Watermelon (Citrullus lanatus L.) represents an economically important cucurbit crop cultivated globally [12]. Nonetheless, it is susceptible to various adverse environmental conditions. Among these, cold stress represents a particularly detrimental factor that markedly diminishes both the yield and quality of watermelon. SMALL AUXIN UP RNA genes (SAURs) are recognized for their critical roles in mediating responses to a range of stressors. However, there remains a limited understanding of the specific functions of watermelon SAURs in relation to abiotic stress tolerance.

In this study, we performed a comprehensive characterization of the SAUR gene ClSAUR1 from watermelon. Quantitative real-time PCR (qRT-PCR) and promoter::β-glucuronidase (GUS) assays demonstrated that the expression of ClSAUR1 was upregulated in response to ABA and chilling treatment. Subsequently, we successfully generated tobacco lines with overexpression of ClSAUR1 and employed these lines to examine photosynthesis, reactive oxygen species (ROS) accumulation, and antioxidant enzyme activities to assess their sensitivity to chilling stress. Additionally, we analyzed the expression profiles of genes associated with ROS, ABA, and cold stress. Functional studies of ClSAUR1 will not only enhance our understanding of the specific roles of SAURs in watermelon adaptation to chilling stress, but may also offer insights into the potential signaling pathways involved in stress responses.

2. Materials and Methods

2.1. Plant Materials and Growth Conditions

We used the watermelon cultivar ‘97103’ for expression analyses. Tobacco plants were utilized for the development of transgenic lines. Watermelon and tobacco plants were cultivated in growth chambers located within greenhouses (16-h light/8-h dark cycle, 65% relative humidity). The environmental conditions included a light intensity of 200 μmol m⁻² s⁻¹. Four-week-old watermelon seedlings were used for treatments involving exogenous ABA and chilling treatments; these treatments involved the seedlings being sprayed with 100 μM of ABA (Sigma, St. Louis, MO, USA) and 4 °C treatment, respectively. The second true leaf on each plant was sampled at time 0 (control), and then again at 1, 4, and 12 h after treatment.

2.2. Identification and Bioinformatics Analysis of ClSAUR1

Initially, we retrieved the sequence of ClSAUR1 from the Cucurbit Genomics Database (accession number: Cla97C01G000145). The conserved domains within the ClSAUR1 protein were identified using SMART. Subsequently, we obtained the protein of ClSAUR1 counterparts from Arabidopsis, tomato, soybean, and cucumber via Phytozome. We performed a phylogenetic analysis using MEGA 5.0 and analyzed the ClSAUR1 promoter with PlantCARE, following prior research [7,13].

2.3. Analysis of Subcellular Localization

ClSAUR1 coding sequences were amplified with gene-specific primers and cloned into the pFGC–eGFP plasmid using BamH I sites (Table S1). The recombinant expression vector was introduced into Agrobacterium tumefaciens and temporarily expressed in tobacco leaf cells. Images were captured 48 h post-transformation.

2.4. GUS Staining Promoter Assay

To examine the expression of ClSAUR1, we utilized a pair of specific primers to amplify a 1500 bp upstream promoter sequence (Table S2). The fragment was ligated into the pBI101 plasmid vector at the Xba I restriction sites, and the resulting construct was designated as ProClSAUR1::GUS (Figure S1). Transgenic tobaccos were generated through the Agrobacterium infection method [14]. Transgenic tobacco seeds were subsequently subjected to screening with 100 mg L⁻¹ kanamycin (KanR) and identified through GUS histochemical staining, employing a GUS Histochemical Staining Kit (O’BioLab, Beijing, China). Subsequently, T3 transgenic lines were screened and harvested for further phenotypic analysis. Seven-day-old tobacco transgenic seedlings derived from the T3 generation and cultivated on 1/2 MS medium were subjected to a treatment at 4 °C. Following a 24-h incubation period, the transgenic seedlings underwent GUS staining and representative digital images were captured using a stereomicroscope.

2.5. Plasmid Construction and Generation of Tobacco Transgenic Plants

The CDS of ClSAUR1 was amplified employing a specific primer pair: OESAUR1-S and OESAUR1-A (Table S3). The PCR-generated amplicons were then subjected to enzymatic digestion and ligation into the pBI121 vector (Figure S1). Following this, the PBI121-P35S::ClSAUR1-GUS construct was introduced into tobacco plants. Transgenic tobacco lines were identified through PCR analysis and GUS histochemical staining, and these lines were subsequently utilized for further experimental investigations (Figure S2, Table S4).

2.6. Chilling Tolerance in Transgenic Tobacco Lines

The T3-generation transgenic lines OESAUR1-1 and OESAUR1-2 and WT seedlings were cultivated on 1/2 MS medium at 4 °C for 7 d. Meanwhile, seeds from the overexpression lines (OESAUR1-1 and OESAUR1-2) as well as WT tobacco were sown in seedling trays and incubated in a growth chamber maintained at 25 °C under 16 h/8 h (light/dark) light cycles. Upon reaching the four-leaf stage, both WT and transgenic tobacco plants were subjected to cold-stress treatment in a growth chamber maintained at 4 °C for a duration of two weeks. Plants cultivated at a standard temperature of 25 °C served as controls. Each experimental condition was replicated three times. Following the completion of the low-temperature treatment, phenotypic observations of the plants were conducted. Additionally, a minimum of three samples from each treatment group were collected, rapidly frozen in liquid nitrogen, and subsequently stored in a −80 °C ultra-low temperature freezer for further analysis of physiological indices and qRT-PCR assessments.

2.7. Determination of Physiological Indices

3, 3′-DAB and nitro blue tetrazolium (NBT) were performed as previously described [15] to assess the levels of hydrogen peroxide (H₂O₂) and O²⁻. The excised tobacco leaves were submerged in DAB (1 mg/mL DAB–HCl, pH 3.8) and NBT solutions (1 mg/mL NBT in 10 mM phosphate buffer with 10 mM sodium azide, pH 7.8). The leaves were subsequently incubated under light at 24 °C for a duration of 6 to 8 h, until the formation of brown or blue precipitates were evident, respectively. The activity of superoxide dismutase (SOD) and peroxidase (POD) was quantified utilizing a commercially available SOD and POD assay kit by quantifying formazan dye levels. The spectrophotometric analysis of superoxide dismutase (SOD) and peroxidase (POD) activities was conducted at wavelengths of 560 nm and 420 nm, respectively (Jiancheng, Nanjing, Jiangsu, China) [16]. The concentrations of malondialdehyde (MDA) and proline were quantified utilizing the Malondialdehyde (MDA) Assay Kit and Proline Assay Kit (Solarbio, Beijing, China), respectively. Fluorescence of chlorophyll was analyzed using chlorophyll fluorometer (PAM 2100, Walz, Effeltrich, Germany).

2.8. Quantitative Real-Time PCR

Total RNA was extracted from both watermelon and tobacco samples. Reverse transcription and qRT–PCR were performed as previously described. The watermelon β-actin and tobacco ACTIN genes were used as reference sequences for designing primers (Table S1) [17].

2.9. Statistical Analysis

Data were analyzed by a two-tailed Student’s t-test or by one-way analysis of variance (ANOVA) using SPSS version 18.0 (IBM, Chicago, IL, United States). Statistical significance was indicated by * or b for p < 0.05, and high significance was denoted by ** or a for p < 0.01.

3. Results

3.1. Isolation and Basic Informatics Analysis of ClSAUR1

The CDS of the ClSAUR1 gene comprised 372 bp, encoding a protein of 123 amino acids. The protein exhibited an isoelectric point (pI) of 123 and a molecular weight of 14.34 kDa. The ClSAUR1 gene was located on chromosome 01 (Chr 01). Furthermore, the ClSAUR1 protein contained a conserved auxin-inducible domain, which it shared with its orthologs. Phylogenetic and sequence similarity analyses further revealed that ClSAUR1 was most similar to AtSAUR32 in Arabidopsis and SlSAUR58 in tomato, which have been studied (Figure 1A). The subcellular localization of the watermelon ClSAUR1 protein was investigated through the transient expression of its GFP fusion constructs in the epidermal cells of tobacco leaves (Figure 1B). The findings indicated that the ClSAUR1 protein is distributed in both the nucleus and the cytoplasm (Figure 1B).

We analyzed the spatial and temporal expression profiles of ClSAUR1 using qRT-PCR. The findings revealed that ClSAUR1 is ubiquitously expressed across various tissues in watermelon, exhibiting the lowest expression levels in the fruit. Notably, ClSAUR1 expression was predominantly observed in root and leaf, with expression levels about 2- and 4-fold higher compared to fruit (Figure 2A).

3.2. Response Patterns of ClSAUR1 to ABA and Chilling Stress in Tobacco Plants

The cis-acting elements within the ClSAUR1 promoter sequence were analyzed using the PlantCARE database. This analysis identified two abscisic acid (ABA) responsive elements (ABREs), two anaerobic responsive elements (AREs), one methyl jasmonate (MeJA) responsive element (TGACG-motif), and one salicylic acid responsive element (TCA-element) (Figure 2B). The expression patterns of ClSAUR1 in response to ABA and chilling stress were detected by qRT-PCR in watermelon leaves at four distinct timepoints (0, 1, 4, and 12 h). The results demonstrated that the ClSAUR1 expression was upregulated 2- and 7-fold 1 to 12 h following ABA treatment, respectively (Figure 2C). Similarly, the expression of ClSAUR1 was induced to 4- and 6-fold at 1 to 4 h, respectively, and then repressed to 3-fold with exposure to chilling treatment (Figure 2D). The ProClSAUR1::GUS analysis indicated an absence of a GUS signal in the roots of 7-day-old seedlings following 24 h exposure to ABA and chilling treatments, as well as in untreated seedlings. Conversely, a significant induction of GUS expression was observed in the leaves and hypocotyl axis in response to 24 h ABA and chilling treatments (Figure 2E,F).

3.3. Overexpression of ClSAUR1 Enhanced Tobacco’s Chilling Stress Tolerance

In this study, we generated transgenic tobacco plants that overexpress ClSAUR1 under the regulation of a CaMV35S promoter. These transgenic lines were subsequently screened using PCR, GUS, and RT-PCR techniques (Figure S2A,B and Figure 3A). To further elucidate the specific roles of ClSAUR1 in response to chilling stress, we subjected 7-day-old seedlings overexpressing ClSAUR1 (OESAUR1-1 and OESAUR1-2 lines), along with WT controls, to a chilling treatment. All experimental plants exhibited wilting, chlorosis, and stunted growth following exposure to a low temperature of 4 °C for a duration of 7 days (Figure 3B). However, the constitutive overexpression of the ClSAUR1 gene in tobacco resulted in a significantly improved condition compared to the WT plants (Figure 3B). Following the investigation of chilling stress, the growth chamber temperature was adjusted to the optimal conditions to facilitate the recovery of the seedlings. This recovery period was maintained for three days to mitigate the effects of chilling stress. Only 4% of the WT plants survived after recovering, whereas 26% to 31% of the OE plants survived (Figure 3B,C). Similarly, four-week-old tobacco plants were subjected to cold treatment at 4 °C. All the plants tested exhibited wilting and chlorosis after being exposed to the low temperature for a duration of two weeks (Figure 3D). However, tobacco with overexpression of the ClSAUR1 gene (OE) also showed significantly better status than the WT plants (Figure 3D). These findings suggest that ClSAUR1 may enhance plant tolerance to chilling stress. The results obtained from the PAM chlorophyll fluorometer indicated a significant phenotypic difference between WT and OESAUR1 seedling leaves under chilling conditions. Specifically, the leaves of OE-1 and OE-2 exhibited higher Fv/Fm levels compared to the WT. In contrast, under normal conditions, there was a negligible difference in Fv/Fm levels between WT and ClSAUR1-overexpressing seedling leaves (Figure 3E,F).

3.4. Alterations in Physiological and Biochemical Indices of Tobacco Overexpressing ClSAUR1 in Response to Chilling Stress

Reactive oxygen species (ROS) are produced in substantial quantities under stress conditions, such as cold stress, and their excessive accumulation can result in oxidative damage to cellular structures [18]. In the current study, DAB and NBT staining assays were employed to assess ROS accumulation in tobacco leaves following exposure to cold treatment. Under standard conditions, the levels of endogenous H₂O₂ and O²⁻ in both WT and ClSAUR1-overexpressing tobacco leaves exhibited no significant differences, as determined by DAB and NBT staining. After exposure to chilling treatment, the concentrations of H₂O₂ and O²⁻ in both WT and OESAUR1 transgenic tobacco leaves increased. However, the OESAUR1 transgenic lines demonstrated reduced staining intensities compared to the WT in both 7-day- and four-week-old seedlings. This observation suggests that the OESAUR1 lines maintain lower levels of H₂O₂ and O²⁻ following chilling stress (Figure 4A–C). The findings revealed no significant differences in the SOD and POD enzyme levels between WT and ClSAUR1-overexpressing tobacco plants under standard conditions. Nevertheless, with the onset of reduced temperatures, an up-regulation of SOD and POD enzyme content was observed in both the WT and OESAUR1 lines (Figure 4D). In addition, MDA content and free proline serve as physiological indicators of stress-induced oxidative damage and osmoregulation, respectively. Consistent patterns were observed for both MDA and proline levels, with significantly elevated concentrations in OESAUR1 compared to the WT upon exposure to chilling stress (Figure 4D). The findings indicate that the overexpression of watermelon ClSAUR1 in tobacco plants enhances tolerance to chilling stress by augmenting reactive oxygen species scavenging, improving osmotic adjustment, and maintaining membrane integrity in the transgenic plants compared to the WT.

3.5. Up-Regulation of Cold Stress-Related Genes in ClSAUR1-Overexpressing Lines Under Conditions of Low-Temperature Stress

To investigate the transcriptional regulatory mechanism of ClSAUR1, the transgenic lines OESAUR1-1 and OESAUR1-2, characterized by elevated expression levels, were utilized to analyze the expression profiles of genes associated with cold response through qRT-PCR (Figure 5). The expression profiles of various genes, encompassing those involved in antioxidant enzyme synthesis (NtPOD and NtSOD), ABA signaling (NtAREB1, NtDREB3, and NtLTP1), and the CBF–COR pathway (NtCOR47, NtCOR78, NtCBF1, and NtCBF2) were examined in ClSAUR1-overexpressing lines subjected to low-temperature treatment (Figure 5). Given that the overexpression of ClSAUR1 has been shown to enhance the antioxidant capacity of tobacco plants under chilling stress, this study quantified the expression levels of genes encoding antioxidant enzymes in ClSAUR1-overexpressing tobacco plants both prior to and following exposure to cold treatment. Under standard growth conditions, the expression levels of NtPOD and NtSOD in ClSAUR1-overexpressing plants exhibited a slight increase compared to WT plants. In contrast, when subjected to chilling stress, these expression levels were markedly upregulated in all transgenic lines relative to the WT (Figure 5). In response to cold stress, the expression levels of three ABA signaling genes, NtABF1, NtABF2, and NtDREB3, were significantly elevated in OESAUR1 plants compared to WT plants (Figure 5). Furthermore, the study examined the impact of ClSAUR1 on cold stress signaling via a CBF-dependent pathway in WT and ClSAUR1-overexpressing (OE) lines under chilling stress conditions. The results indicated that there were no significant differences in the expression levels of the genes among all lines under normal conditions at 25 °C. However, upon exposure to chilling stress at 4 °C, the expression of these genes was upregulated, with significantly higher levels observed in the OE lines compared to the WT. These findings indicate that ClSAUR1 enhances plant tolerance to low-temperature stress through the modulation of antioxidant, abscisic acid (ABA) signaling, and CBF-dependent pathways.

4. Discussion

At present, there is a paucity of research regarding the involvement of SAUR proteins in plant responses to chilling stress. In this study, our findings indicate that ClSAUR1 expression is significantly upregulated in response to ABA and cold treatment (Figure 2B–F). ClSAUR1 is most similar to Arabidopsis AtSAUR32 and tomato SlSAUR58, which are evolutionarily conserved and have been demonstrated to participate in ABA signaling and drought stress responses [7,19]. Prior research has established that the subcellular localization of SAUR proteins exhibits considerable diversity. Our study determined that the ClSAUR1 protein is concurrently localized within the nucleus and cytoplasm of tobacco leaves (Figure 1B), aligning with the subcellular localization patterns observed for AtSAUR32 and SlSAUR58. Subsequently, we developed transgenic tobacco lines with overexpression of ClSAUR1. These transgenic lines exhibited reduced wilting, decreased leaf yellowing, and significantly improved overall condition compared to WT plants under chilling stress conditions. Therefore, our findings suggest that the overexpression of ClSAUR1 enhances the chilling tolerance in tobacco (Figure 3 and Figure 4).

During the process of cold stress signal transduction, ROS function as secondary messengers, initiating a cascade of stress responses [20]. However, the detrimental effects associated with the excessive accumulation of ROS induced by cold stress have also been documented [21]. In this study, it was observed that tobacco lines overexpressing ClSAUR1 exhibited reduced ROS accumulation compared to WT plants following cold treatment, as evidenced by DAB and NBT staining (Figure 4A–C). Furthermore, the overexpression of the ClSAUR1 gene in transgenic tobacco plants was associated with enhanced POD and SOD activities under low-temperature conditions (Figure 4D). These enzymes alleviate the impact of chilling stress through the scavenging of ROS [22]. In this study, two representative ROS scavenger genes, NtSOD and NtPOD, were selected for transcript level analysis. The expression of these genes was found to be up-regulated under chilling stress, with a more pronounced up-regulation observed in overexpression (OE) lines (Figure 5). Previous studies have demonstrated that the up-regulation of the ROS detoxification system at the transcriptional level can substantially improve plant survival under low-temperature conditions [23]. Additionally, the OE tobacco plants exhibited higher accumulations of proline and MDA compared to the control (Figure 4D), indicating that the OE plants experienced less severe damage from ROS accumulation, since proline and MDA are the products of membrane lipid peroxidation and membrane injury [24,25]. The aforementioned studies demonstrated that the overexpression of ClSAUR1 significantly increased proline and MDA content, as well as the activity of antioxidant enzymes, while concurrently reducing ROS release and alleviating the adverse effects of low temperatures on plants.

The present study suggests that ABA signaling pathways play a role in stress responses targeting the watermelon gene ClSAUR1. Notably, two ABA-responsive elements have been identified within the promoters of ClSAUR1 (Figure 2B). qRT-PCR and promoter::GUS staining analyses demonstrated a significant induction of ClSAUR1 expression in response to exogenous ABA treatment (Figure 2D,F). Additionally, under chilling stress conditions, the transcript levels of ABA signaling genes, namely NtABF1, NtABF2, and NtDREB3, were markedly elevated in plants overexpressing ClSAUR1 (Figure 5). The findings indicate that the enhanced cold tolerance observed in ClSAUR1-overexpressing plants may be attributed to ABA signaling and the expression of cold-responsive genes. It has been demonstrated that numerous SAURs can regulate plant stress resistance via ABA-mediated pathways. For instance, certain SAUR genes have shown a clear response to ABA treatment [7,26]. Arabidopsis AtSAUR41, AtSAUR32, and poplar PtSAUR8 enhance plant drought resistance via ABA-mediated pathways [6,7,8]. Our findings suggest that watermelon ClSAUR1 may influence ABA signaling to modulate cold responses, activating defense and chilling stress-responsive genes in tobacco.

Exposure to chilling stress swiftly activates the expression of various transcription factors, notably the AP2 family CBFs, which subsequently induce a multitude of downstream cold-responsive (COR) genes, thereby enhancing chilling tolerance [27,28]. The transcriptional cascade of CBF–COR constitutes the primary regulatory framework within the cold signaling pathway and serves as a critical determinant of plant freezing tolerance [29]. This study found that ClSAUR1 overexpression increased the levels of the cold-responsive genes NtCBF1, NtCBF2, NtCOR47, and NtCOR78 during chilling stress (Figure 5). Based on the experimental results, ClSAUR1 positively regulates chilling stress via the CBF–COR pathway, making it a cold-responsive SAUR that enhances plant cold tolerance. The overexpression of ClSAUR1 augments the ROS detoxification system and the biosynthesis of osmoprotectants in plants subjected to low-temperature conditions. Additionally, ClSAUR1 is involved in the transcriptional regulation responsive to cold stress through the ROS, ABA, and CBF–COR pathways (Figure 6). Our results suggested that ClSAUR1 functions as a positive regulator in the adaptation of plants to low-temperature conditions.

5. Conclusions

Chilling stress represents a substantial threat to the growth and productivity of watermelon. SAUR genes are integral to developmental processes and stress responses. In this study, we identified a SAUR gene in watermelon, designated as ClSAUR1, and elucidated its critical role in the plant’s response to chilling stress. Through qRT-PCR and promoter::GUS analyses, we established that the expression of ClSAUR1 in tobacco is inducible by exogenous abscisic acid (ABA) and chilling stress. The overexpression of ClSAUR1 enhances tolerance to chilling stress in tobacco plants. Tobacco with ClSAUR1 overexpression exhibited reduced reactive oxygen species (ROS) levels and elevated activities of antioxidant enzymes when subjected to chilling stress. qRT-PCR analysis indicated a significant up-regulation of specific marker genes associated with ROS, ABA, and cold response pathways. In conclusion, ClSAUR1 appears to enhance plant tolerance to cold stress by modulating antioxidant capacity and regulating the expression of genes associated with defense mechanisms, cold response, and ABA signaling pathways (Figure 6). We present evidence substantiating the role of ClSAUR1 in the response to cold stress, investigate the underlying molecular mechanisms, and offer a valuable resource for the potential application of ClSAUR1 in the genetic enhancement of chilling stress tolerance in crops.