BnaMYB73, a Brassica napus L. R2R3-MYB Transcription Factor, Enhances Plant Salt and Osmotic Stress Tolerance

TL;DR

This study identifies BnaMYB73, a gene in rapeseed, that helps plants tolerate salt and osmotic stress without affecting normal growth.

Contribution

BnaMYB73 is newly characterized as a stress-responsive R2R3-MYB transcription factor in Brassica napus.

Findings

BnaMYB73 expression increases under salt and osmotic stress.

Transgenic Arabidopsis with BnaMYB73 showed higher stress tolerance and improved physiological responses.

Stress-related genes like AtRD29B and AtSOS1 were upregulated in BnaMYB73-expressing plants.

Abstract

MYB transcription factors (TFs) are crucial for plant growth, development, and response to abiotic stress. However, their exact functions in abiotic stress responses in rapeseed remain largely unexplored. In this study, we identified and characterized BnaMYB73, a member of the R2R3-MYB subfamily, and investigated its role in abiotic stress tolerance. The transcription level of BnaMYB73 was significantly upregulated in response to salt and osmotic stress. Transgenic Arabidopsis thaliana lines expressing BnaMYB73 displayed significantly enhanced tolerance to salt and osmotic stress, while showing no phenotypic differences in growth compared with wild-type (WT) plants under normal conditions. Physiological analyses revealed that the BnaMYB73-expressing plants accumulated higher proline levels, exhibited elevated superoxide dismutase (SOD) and peroxidase (POD) activities, and reduced malondialdehyde (MDA) content under stress conditions. Moreover, the BnaMYB73-expressing plants significantly upregulated key stress-responsive genes, including AtRD29B, AtDREB2A, AtRAB18, AtP5CS1, AtSOS1 and AtCAT1. Collectively, these findings establish BnaMYB73 functions as a stress-responsive transcription factor that enhances abiotic stress tolerance and provide a promising target for breeding stress-resilient rapeseed cultivars.

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1. Introduction

Adverse external environments, such as salt and drought, severely inhibit plant growth and development, leading to lower yields [1]. To cope, plants have evolved efficient regulatory mechanisms [2]. Proline accumulation during stress helps regulate osmotic balance and reduces reactive oxygen species (ROS)-induced oxidative damage [3,4]. Additionally, stress-responsive genes, including SOS1, P5CS1 and CAT1, are activated to improve stress tolerance [5,6,7]. Key transcription factors (TFs) like MYB, WRKY, NAC and AP2/ERF mediate these responses through binding to these gene promoter cis-elements [8,9,10,11].

MYB TFs are prevalent and essential, controlling growth, development, and stress responses via interaction with Myb cis-acting elements of target gene promoters [10]. Their conserved DNA-binding domain contains one to four repeats (each ~52 amino acids), folding into three α-helices, with the second and third helices forming a helix-turn-helix structure [12]. Repeats are designated R1, R2, and R3 based on their homology with the c-Myb protein [12]. According to the repeats, MYB are classified as MYB-related, R2R3, R1R2R3, and 4R, with R2R3 MYBs being the most abundant [12].

It is well documented that R2R3 MYB members have essential functions in plant growth, development, and abiotic stress responses. In Arabidopsis, AtMYB20 is upregulated by salt and drought, and its overexpression enhances salt tolerance through suppression of PP2Cs expression [13]. Similarly, AtMYB21 overexpression increases salt tolerance during both germination and vegetative growth in Arabidopsis [14]. AtMYB49 improves salt tolerance in an ABA-dependent manner by regulating cuticle biosynthesis and antioxidant defense [15]. AtMYB68 enhances drought resistance and ABA sensitivity during germination and seedling growth. Correspondingly, transgenic canola plants expressing AtMYB68 display superior tolerance to diverse abiotic stresses, accompanied by higher yield performance [16]. AtMYB93 negatively regulates lateral root formation through interacting with ARABIDILLO and promoting its degradation [17]. In rice, OsMYB2 is induced with salinity, drought, and low temperature, and transgenic lines exhibit enhanced tolerance and increased ABA sensitivity due to increased osmolyte accumulation, antioxidant activity, and reduced oxidative damage [18]. In tomato, SlMYB49 enhances resistance to Phytophthora infestans, leading to reduced necrosis, smaller lesion size, lower pathogen load, and decreased disease severity [19]. In alfalfa, MsMYB4 expressed in Arabidopsis markedly improves salt tolerance in an ABA-dependent manner, as evidenced by higher germination rates and increased root elongation [20].

Different from the one described above, MYB73 exhibits dual regulatory behavior, acting positively or negatively depending on plant species. In Arabidopsis, AtMYB73 acts as a negative regulator, as evidenced by the greater salt tolerance of atmyb73 mutants relative to WT plants [21]. Similarly, GmMyb73, mainly expressed in the root, suppresses tolerance to low-phosphorus stress in soybean [22]. Contrary to its negative regulatory activity, MYB73 acts as a positive regulator in wheat and cotton. In wheat, TaMYB73 expression is induced by various abiotic stresses, including salinity, drought, and hormonal signals. Arabidopsis lines overexpressing TaMYB73 display greater tolerance to NaCl, LiCl, and KCl, as well as enhanced germination under salt and ABA stress [23]. In cotton, GhMYB73, induced by salt and ABA, improves salt resistance, while its silencing increases NaCl sensitivity [24].

Rapeseed (Brassica napus L.) is a widely cultivated oil crop. However, its growth and yield are frequently constrained by adverse environmental factors, particularly salinity and drought [25,26]. Despite extensive research on MYB73 in various plants, its biological functions in stress regulation in oilseed crops remain largely uncharacterized. In this work, we cloned the rapeseed BnaMYB73 and elucidated its role in salt and drought stress responses. BnaMYB73 expression was upregulated by salt and osmotic stress, and heterologous expression of BnaMYB73 in Arabidopsis conferred elevated salt and drought resilience, identifying it as a positive modulator in abiotic stress responses.

2. Materials and Methods

2.1. Multiple Sequence Alignment, Phylogenetic Analysis and Three-Dimensional Structure Prediction

MYB protein sequences from Brassica napus, Arabidopsis thaliana, Gossypium hirsutum, and Glycine max were aligned using the ClustalW program v2.0.11. A Neighbor-Joining phylogenetic tree was generated to elucidate the evolutionary relationships among these sequences, with 1000 bootstrap replications performed in MEGA 11.0. The three-dimensional structure of BnaMYB73 was predicted using the SWISS-MODEL server and visualized with Pymol software v3.1.

2.2. Rapeseed Growth Environments and Stress Treatments

The Brassica napus cultivar ‘Zhongshuang 11’ and Arabidopsis thaliana ecotype Col-0 were used in this study. Plant growth conditions followed previously reported protocols [27]. Seeds were surface-sterilized with 75% (v/v) ethanol and rinsed five times with sterile ddH_2_O. Sterilized seeds were germinated on half-strength Murashige and Skoog (1/2 MS) medium and grown under controlled conditions (24 °C/22 °C, 50–60% relative humidity, 16 h light/8 h dark photoperiod). After one week, seedlings were transplanted to a 1:1 (v/v) mixture of vermiculite and nutrient soil and cultivated in a greenhouse. For tissue-specific expression analysis, roots, stems, leaves, flowers, and siliques were collected from six-month-old rapeseed plants. For stress treatments, five-week-old rapeseed plants were treated with either 150 mM NaCl or 20% (w/v) PEG6000 for 0 h, 1 h, 3 h, 6 h, 9 h, 12 h and 24 h, after which leaf and root samples were harvested for further experiments. Two-week-old Arabidopsis seedlings, including wild-type and transgenic lines, were exposed to 200 mM NaCl or 300 mM mannitol for 9 h, followed by leaf sampling for RNA extraction and physiological measurements.

2.3. Transactivation Analysis of BnaMYB73 in Yeast Cells

The full-length coding sequence of BnaMYB73 was amplified using gene-specific primers listed in Table S1 and cloned into the pGBKT7 vector to generate the recombinant plasmid pGBKT7-BnaMYB73. This resulting plasmid was then introduced into the yeast strain AH109. Yeast cells transformed with the empty pGBKT7 vector and pGBKT7-VP16 served as negative and positive controls, respectively. Yeast transformants were first selected on SD/-Trp medium and subsequently replicated onto SD/-Trp/-His medium supplemented with 4 mg/mL X-α-gal (SD/-Trp/-His/X). After incubation for three days, the transactivation activity of BnaMYB73 was assessed based on yeast phenotypes.

2.4. Subcellular Localization of BnaMYB73

To determine the subcellular localization, the coding sequence of BnaMYB73 was fused to the pGreen-35S-GFP vector (35S-GFP), resulting in the 35S:BnaMYB73-GFP construct. This recombinant plasmid was introduced into Agrobacterium tumefaciens GV3101 and transiently expressed in Nicotiana benthamiana leaves as previously described [28]. For co-localization analysis, transgenic tobacco plants expressing a nuclear-localized red fluorescent protein (NLS-mCherry) were used as a nuclear marker [29]. Forty-eight hours post-injection, fluorescence signals were observed using laser scanning confocal microscopy (Zeiss LSM710, Jena, Germany). GFP was excited at 488 nm with emission collected between 500 and 540 nm, while mCherry was excited at 559 nm with emission collected between 600 and 680 nm.

2.5. Quantitative Real-Time PCR Experiment

The collected samples were homogenized for RNA extraction and subsequent RT-qPCR analysis. Total RNA was isolated and reverse-transcribed into cDNA using the Vazyme R423-01 kit (Vazyme Biotech Co., Ltd., Nanjing, China) according to the manufacturer’s instructions. BnaActin from rapeseed and AtActin from Arabidopsis were used as the reference genes. Primers used to perform RT-qPCR are listed in Table S1. The 2^−ΔΔCt^ method established by Livak and Schmittgen [30] was used to quantify relative gene expression.

2.6. Stress Tolerance Experiments of the Transgenic Yeast

The coding sequence of rapeseed BnaMYB73 was inserted into the yeast expression vector pYES2 (Coolaber (Beijing, China), PL001) and transformed into yeast strain INVSc1 (Coolaber, CC303). Yeast carrying either pYES2-BnaMYB73 or the empty vector was initially cultured in SC-ura liquid medium supplemented with 2% glucose at 30 °C for 24 h. Subsequently, cultures were then diluted to an OD_600_ of 0.4 in induction medium (SC-ura liquid medium with 2% galactose) and incubated for 36 h, after which cell densities were adjusted to OD_600_ = 1.8. For assessing salt and osmotic stress tolerance, the transformed yeasts were cultured in 4 M NaCl or 1 M mannitol for 48 h in an incubator, with sterile ddH_2_O as a control.

2.7. Plant Genetic Generation

To create BnaMYB73-expressing Arabidopsis, the coding sequence was amplified and inserted into the binary vector pCAMBIA2301 under the CaMV35S promoter using Vazyme ClonExpress II One Step Cloning Kit. The resulting construct was introduced into GV3101 for subsequent Arabidopsis transformation according to the established protocol [31]. After transformation, the harvested seeds were germinated on 1/2 MS medium with 50 µg/mL kanamycin [32]. Kanamycin-resistant seedlings were transplanted to soil and cultivated to maturity. Homozygous lines were confirmed through antibiotic resistance segregation analysis in the subsequent generations. Expression verification of BnaMYB73 was performed using RT-qPCR on eight independently derived overexpression lines.

2.8. Germination Assay

For germination assays, fifty surface-sterilized seeds from each genotype were sown on 1/2 MS medium supplemented with either 100 mM NaCl, 250 mM mannitol, or without additives as the control. Plates were incubated under controlled growth conditions. Seed germination was monitored daily for 7 days and was defined by radicle emergence through the seed coat. Additionally, cotyledon greening rate, defined as the percentage of seedlings with fully expanded green cotyledons, was also assessed. All experiments were performed with three biological replicates.

2.9. Stress Tolerance Assays

To assess stress tolerance at the seedling stage, three-day-old Arabidopsis thaliana seedlings were transplanted to 1/2 MS medium supplemented with either 100 mM NaCl, 300 mM mannitol, or without additives as the control. Seedling growth was monitored daily, and after seven days of treatment, growth performance was recorded. Growth parameters included fresh biomass and primary root length, and the root length inhibition rate was calculated to quantify the effects of salt and osmotic stress. For stress tolerance assays at the adult plant stage, ten-day-old seedlings were transplanted into pots containing a 1:1 (v/v) mixture of vermiculite and nutrient soil and grown under controlled environmental conditions for three weeks. Thereafter, salt stress was imposed by irrigating with 300 mM NaCl for seven days, while drought stress was induced by withholding irrigation for three weeks. During the stress treatments, plant growth parameters and phenotypic responses were systematically recorded to assess stress tolerance.

2.10. Physiological and Gene Expression Analysis

Fourteen-day-old WT and BnaMYB73-expressing lines were exposed to 200 mM NaCl or 300 mM mannitol for nine hours under controlled growth conditions. Following stress exposure, leaves were harvested to evaluate a range of physiological parameters, including superoxide dismutase (SOD) and peroxidase (POD) activities, malondialdehyde (MDA) and proline contents. All measurements were conducted using standardized assay kits (Solarbio, Beijing, China; BC5165 for SOD activity; BC0090 for POD activity; BC0025 for MDA content; BC0290 for proline content). Concurrently, total RNA was extracted from treated leaves for RT-qPCR to determine the transcript levels of stress-responsive genes: RD29B, DREB2A, RAB18, P5CS1, SOS1, and CAT1. Gene expression was normalized to internal reference genes AtActin (AT2G37620) and BnaActin (FJ529167.1) using the 2^–ΔΔCt^ method. The corresponding accession numbers for the analyzed genes and proteins are as follows: BnaMYB73 (XP_013686616.1), AtMYB73 (AT4G37260; NP_195443.1), GhMYB73 (XP_016709948.2), GmMYB73 (Glyma.01G190100.1), RD29B (AT5G52300), DREB2A (AT5G05410), RAB18 (AT5G66400), P5CS1 (AT2G39800), SOS1 (AT2G01980), CAT1 (AT1G20630), AtActin (AT2G37620), and BnaActin (FJ529167.1).

2.11. Statistical Analysis

All data, expressed as means ± SD, are from three independent biological replicates, each consisting of at least 10 plants to ensure accuracy and reproducibility. Statistical significance was established using Student’s t-test, with p-value less than 0.05 (*) and 0.01 (**).

3. Results

3.1. BnaMYB73 Encodes an R2R3 MYB Transcription Factor in Rapeseed

To characterize the functional role of BnaMYB73 in rapeseed, the gene was cloned and analyzed using multiple sequence alignment, phylogenetic relationship, and 3D structure analysis. The BnaMYB73 coding sequence spans 915 bp and encodes 304 amino acids. Sequence analysis revealed two highly conserved R2 and R3 repeats at the N-terminal region, classifying it within the R2R3-MYB subfamily (Figure 1A). Further phylogenetic analysis revealed that BnaMYB73 exhibited the highest sequence similarity with the Arabidopsis thaliana AtMYB73, sharing 79.63% amino acid identity (Figure 1B). Moreover, the 3D structure model indicated that the R2 and R3 repeats domain contains three α-helices, where the second and third helices form a characteristic helix-turn-helix structure (Figure 1C).

3.2. BnaMYB73 Is a Nuclear-Localized Transcriptional Activator

To investigate the subcellular localization of BnaMYB73, 35S:BnaMYB73-GFP or 35S:GFP constructs were transiently expressed in Nicotiana benthamiana leaves, together with the nuclear marker NLS-mCherry. Overlapping GFP and mCherry signals revealed that rapeseed BnaMYB73 is predominantly localized in the nucleus (Figure 2A). To assess its transcriptional activation, pGBKT7-BnaMYB73, pGBKT7-VP16, or the empty pGBKT7 vector was transformed into the yeast strain AH109. All transformants grew on SD/-Trp medium, confirming successful transformation. Notably, yeast cells carrying pGBKT7-BnaMYB73 and pGBKT7-VP16 turned blue on SD/-Trp/-His/X medium, suggesting that BnaMYB73 can activate the MEL1 reporter gene and functions as a transcriptional activator (Figure 2B).

3.3. BnaMYB73 Expression Is Upregulated in Response to Salt and Osmotic Stress in Rapeseed

To investigate the tissue-specific expression, BnaMYB73 transcript levels were quantified in various tissues and organs, including silique, root, stem, leaf, and flower, from six-month-old rapeseed plants. The results indicated rapeseed BnaMYB73 is widely expressed across all examined tissues, with the highest abundance detected in roots (Figure 2C). We further investigated the BnaMYB73 expression in five-week-old rapeseed plants treated with 150 mM NaCl or 20% PEG6000. Under both salt and osmotic stress, BnaMYB73 expression was upregulated in leaves and roots, suggesting a potential role in mediating responses to abiotic stress (Figure 2D,E).

3.4. BnaMYB73 Enhances Salt and Osmotic Tolerance in Yeast

To preliminarily investigate the functional roles of BnaMYB73 in eukaryotic systems, the gene was inserted into the yeast expression vectors pYES2 and transformed into the yeast strain INVSc1. Transformed yeast harboring either pYES2-BnaMYB73 or the empty vector was exposed to ddH_2_O, 4 M NaCl, or 1 M mannitol for 48 h. However, under normal conditions, all yeast strains displayed similar growth phenotypes. However, under salt and osmotic stress, yeast cells carrying pYES2-BnaMYB73 exhibited significantly enhanced growth compared to those carrying the empty vector, suggesting increased tolerance to both salt and osmotic stress (Figure S1).

3.5. BnaMYB73 Expression Enhances Seed Germination in Response to Salt and Osmotic Stress

To investigate the biological role of BnaMYB73 in response to salt and osmotic stress, eight homozygous Arabidopsis lines expressing BnaMYB73 were generated and their expression levels were confirmed via RT-qPCR. Three independent transgenic lines (OE#3, OE#4, and OE#7), representing high, medium, and low expression levels, were selected for further analysis (Figure S2). Seed germination and cotyledon greening rates were evaluated through sowing WT and BnaMYB73-expressing seeds on 1/2 MS medium supplemented with 0, 100 mM NaCl, or 250 mM mannitol for 7 days (Figure 3A). Under normal growth conditions, germination and cotyledon greening rates were comparable between WT and transgenic seeds (Figure 3B,E). In contrast, under salt and osmotic stress conditions, both germination rate and cotyledon greening rate were significantly reduced. Notably, OE#3 and OE#4 seeds showed significantly higher germination and cotyledon greening rates compared to WT and OE#7 seeds, although their germination rate became comparable after 7 days (Figure 3C–E). In conclusion, BnaMYB73-expression Arabidopsis lines led to enhanced seed germination and cotyledon development under NaCl and osmotic stress, indicating improved salt and osmotic stress tolerance during early seedling development.

3.6. BnaMYB73 Expression Improves Salt and Drought Tolerance in Arabidopsis Seedlings and Adult Plants

We then examined the effects of salt and osmotic stress on the growth of transgenic Arabidopsis at the seedling stage. Under control conditions, all lines displayed comparable growth and morphology (Figure 4A,D,E). Upon exposure to 100 mM NaCl or 300 mM mannitol conditions, growth was suppressed in every line, with the transgenic seedlings exhibiting significantly greater root length, biomass, and reduced root length inhibition relative to WT (Figure 4B–F). These results indicate that BnaMYB73 expression in Arabidopsis enhances salt and osmotic tolerance at the seedling stage.

To further evaluate the effect of abiotic stress on the growth of mature plants, seedlings were transplanted into greenhouse soil and subjected to 300 mM NaCl for one week or drought stress via withholding water for three weeks. Under normal conditions, both WT and transgenic lines exhibited similar survival rates, plant heights, and overall growth (Figure 5A,D,F). Following stress treatments, transgenic plants outperformed WT plants, exhibiting higher survival, greater plant height, lower growth inhibition and water loss rate (Figure 5B–G). Collectively, these findings suggest that expression of BnaMYB73 enhances salt and drought tolerance in adult plants.

3.7. BnaMYB73 Expression Increases Antioxidant Capacity and Reduces Lipid Peroxidation in Transgenic Arabidopsis

Given that salt and drought can induce osmotic stress in plants, we further investigated whether BnaMYB73 expression could enhance oxidative stress resistance in transgenic plants. To assess this, key physiological parameters, such as SOD activity, POD activity, proline content, and MDA content, were quantified in both WT and BnaMYB73-expressing lines. Under normal conditions, no significant differences were observed between WT and transgenic lines. However, under salt or osmotic stress, all lines showed elevated SOD and POD activities, as well as increased proline and MDA levels. Notably, compared to WT, transgenic Arabidopsis exhibited significantly higher SOD and POD activities, greater proline accumulation, and notably lower MDA levels than WT (Figure 6). These findings imply that BnaMYB73 promotes ROS scavenging and alleviates membrane lipid peroxidation, thereby strengthening the plants’ tolerance to abiotic stress.

3.8. Expression of BnaMYB73 Upregulates Stress-Responsive Genes in Transgenic Arabidopsis

To elucidate how BnaMYB73 enhances abiotic stress tolerance, we detected the expression levels of six well-characterized stress-responsive genes (RD29B, DREB2A, RAB18, P5CS1, SOS1, and CAT1) in both WT and BnaMYB73-expressing Arabidopsis under normal and stress conditions using RT-qPCR. Under normal conditions, the transcript levels of all six genes were relatively low, with no significant differences between WT and transgenic lines (Figure 7). However, upon exposure to salt or osmotic stress, the expression of RD29B, DREB2A, RAB18, P5CS1, SOS1, and CAT1 was significantly upregulated in all Arabidopsis lines. Specifically, transgenic lines exhibited substantially higher expression accumulation of all six genes compared to WT. This pronounced upregulation correlates with the enhanced tolerance to abiotic stress observed in BnaMYB73-expressing plants (Figure 7). These findings strongly indicate that BnaMYB73 enhances salt and osmotic stress tolerance via activating a network of stress-responsive genes involved in osmotic regulation, antioxidant defense, and ion homeostasis.

4. Discussion

Adverse environments impose serious constraints on crop growth, development and yield. Numerous studies have emphasized the pivotal functions of TFs in regulating downstream stress-responsive gene expression to mitigate abiotic stress effects. Among them, R2R3 MYB TFs, the most abundant MYB family members in plants, are key regulators in governing plant growth, developmental processes, and adaptation to abiotic stress [12]. Rapeseed, an important oil crop widely used for consumption and oil extraction, has substantial economic value and ecological benefits. However, it is highly susceptible to environmental stressors, leading to poor quality and decreased production [25,26]. Previous analysis has revealed that there are 249 R2R3 MYB TFs in the B. napus genome, yet their roles in abiotic stress responses remain largely unexplored [33]. In this work, we isolated and characterized an R2R3 MYB gene, BnaMYB73. Previous research has shown that MYB proteins with sequence similarity typically share similar functions [34]. The investigation into BnaMYB73 not only helps to elucidate its role in the abiotic stress response of rapeseed but also contributes to a broader comprehension of MYB TFs in other plants. Despite the divergence in full-length sequences, the MYB domains exhibited remarkable conservation among these homologs (Figure 1). The differences in the full-length sequences, coupled with the conserved MYB domain, suggested that MYB proteins retained core functions across plant species, while acquiring specific functions during evolution.

Numerous studies have revealed that the majority of transcription factor-encoding genes are induced by abiotic stresses, and their overexpression in plants has emerged as a useful approach to enhance crop tolerance to such stressors [35,36,37,38,39]. In this study, RT-qPCR indicated a significant upregulation of BnaMYB73 expression in rapeseed under various stress conditions (Figure 2). Furthermore, we generated transgenic Arabidopsis overexpressing the BnaMYB73 gene to assess its role in the abiotic stress response. Under stress conditions (salt and osmotic stress), these transgenic plants exhibited faster germination and improved growth compared to WT, consistent with the functions reported for other MYB genes, such as AtMYB49 [15], GhMYB73 [24], OsMYB2 [18], and TaMYB19 [40] (Figure 3, Figure 4 and Figure 5). These results suggested that overexpression of BnaMYB73 in Arabidopsis enhanced plant growth under salt and drought, confirming that BnaMYB73 elevated stress tolerance at different developmental stages. In summary, our findings implied a positive role of BnaMYB73 in regulating salt and drought tolerance.

To adapt to unfavorable conditions, plants undergo rapid physiological alterations [41,42]. Hence, measuring physiological indicators related to plant stress response can evaluate plant stress resistance. In this study, we assessed four physiological parameters, SOD activity, POD activity, proline content and MDA content (Figure 6). Our study revealed a significant increase in proline content in BnaMYB73-expressing plants compared to WT under all stress conditions, suggesting that the accumulated proline contributed to the enhanced stress tolerance in the transgenic lines. The reduced MDA content in BnaMYB73-expressing plants compared to WT suggested that BnaMYB73 could improve stress tolerance by alleviating lipid peroxidation and maintaining osmotic homeostasis. While changes in physiological indicators alone may not fully alleviate the damages caused by abiotic stress, they are undoubtedly key factors in plant resistance to adverse conditions. Collectively, the physiological changes in BnaMYB73-expressing plants promoted their adaptation to an adverse environment.

The overexpression of BnaMYB73 led to the upregulation of six genes, RD29B, DREB2A, RAB18, P5CS1, SOS1 and CAT1 under abiotic stresses, thereby enhancing the stress tolerance of BnaMYB73-expressing Arabidopsis lines (Figure 7). In transgenic lines, the increased expression of P5CS1 resulted in proline accumulation, which helped regulate osmotic pressure. Additionally, relative to WT, the elevated expression level of SOS1 in BnaMYB73-overexpressing lines under salt and osmotic stress alleviated the cellular toxicity induced by Na^+^. The upregulation of CAT1 in these plants facilitated the removal of excess ROS, further enhancing abiotic stress resilience. While the heterologous overexpression in Arabidopsis provides robust initial evidence for BnaMYB73’s positive regulatory role in abiotic stress tolerance, we acknowledge that a significant amount of work remains to be done to fully understand the regulatory mechanisms by which BnaMYB73 elevates abiotic stress resilience. First, the functional validation relies heavily on a heterologous system in this study, and the physiological relevance within the rapeseed remains to be fully established. Second, the proposed regulatory mechanism, while supported by correlative expression data, lacks direct molecular evidence for BnaMYB73 binding to the promoters of downstream stress-responsive genes. To build upon these findings and address these gaps, our future work will focus on the following: (1) generating and characterizing both BnaMYB73-overexpressing and CRISPR/Cas9-mediated knockout lines in rapeseed to confirm its function in the native genetic background and assess its potential for crop improvement; and (2) employing chromatin immunoprecipitation sequencing (ChIP-seq) in conjunction with Dual-LUC to identify its direct target genes, thereby elucidating the precise transcriptional network it governs under stress conditions.

5. Conclusions

In conclusion, we cloned an R2R3 MYB gene, BnaMYB73, from rapeseed and identified its role in the abiotic stress response. To extensively characterize BnaMYB73, we examined its expression patterns, phenotypic analysis of transgenic plants, and its mechanistic study. Expression examinations disclosed that BnaMYB73 was responsive to salt and osmotic stress. The differences in germination and growth between WT and BnaMYB73-expressing plants indicated that BnaMYB73 expression enhanced tolerance to abiotic stress. Additionally, constitutive expression of BnaMYB73 also improved antioxidant defense to confront abiotic stress. The above findings elucidate the role of BnaMYB73 in stress adaptation and position it as a promising candidate for advancing stress-tolerant rapeseed breeding.