Functional study of the AfRAP2 gene in Amorpha fruticosa L. tolerance to saline-alkali and drought stress

Abstract

Background

Amorpha fruticosa L. is a leguminous shrub with high tolerance to drought, poor soil, and saline-alkali stress conditions. As a member of the family of transcription factors in higher plants, the ethylene response factor AP2/ERF plays a crucial role in both plant adaptation to abiotic stress and in growth and development. In this study, based on genes identified from the transcriptomic sequencing of Amorpha fruticosa L. under drought stress, the upregulated gene AfRAP2 was isolated from its seedlings, with the aim of elucidating its stress-response function using molecular biological techniques.

Results

In this study, the AfRAP2 gene was cloned from the leaves of Amorpha fruticosa L. using RT-PCR. Bioinformatics analysis revealed that AfRAP2 contains an AP2 domain and belongs to the DREB subfamily of the AP2/ERF transcription factor family, showing close phylogenetic relationships with LaEREBP from Lathyrus albus. Real-time quantitative PCR (RT-qPCR) results indicate that AfRAP2 is expressed in various tissues of Amorpha fruticosa L., with the highest expression in leaves and the lowest in stems, furthermore, its expression is significantly upregulated in roots and leaves upon induction by NaHCO₃ and PEG6000. Subcellular localization experiments confirmed that the AfRAP2 protein is localized to the nucleus, and GUS histochemical staining assay revealed that its promoter drives GUS expression in anthers. Resistance analysis of overexpressing yeast strains showed that yeast transformed with the AfRAP2 gene exhibited significantly better growth under sorbitol, mannitol, and NaHCO₃ stress conditions compared to the control, indicating that this gene enhances yeast tolerance to drought and saline-alkali stress. We screened transgenic tobacco and Populus davidiana × P. alba var. Pyramidalis. The results showed that under natural drought and saline-alkali stress treatments, the transgenic lines exhibited significantly improved growth and higher activities of the physiological indicators of catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD) compared with wild-type plants, indicating that the overexpression of the AfRAP2 gene plays a key role in the response to saline-alkali stress and drought stress.

Conclusion

In summary, AfRAP2 contains an AP2 domain and belongs to the DREB subfamily of transcription factors, under abiotic stress induced by NaHCO₃ and mannitol, it can induce the expression of the AfRAP2 gene in tobacco and Populus davidiana×P. alba var. pyramidalis. AfRAP2 plays a vital role in the plant response to saline-alkali stress and drought stress and is a promising candidate gene for stress-tolerant breeding.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s12870-026-09778-y.

Introduction

Amorpha fruticosa L. is a deciduous perennial shrub belonging to the Fabaceae family and native to North America [1, 2], it was introduced to China as an ornamental plant [3]. Amorpha fruticosa L. is characterized by its exceptional stress tolerance, including drought, cold, and saline-alkali stress conditions [4]. Amorpha fruticosa L. has high biomass in its stems and leaves, and its leaves serve as high-protein green forage. It holds significant value in livestock production in arid and semi-arid regions and possesses strong windbreak, sand fixation, and soil and water conservation functions. It is a commonly used pioneer plant in vegetation restoration and ecological management in the arid and semi-arid regions of Northwest China [5]. Studies have shown that under drought stress simulated by PEG6000, the number of germinating seeds and the germination rate of Amorpha fruticosa L. were not significantly different from those in hydroponic conditions. This indicates that drought has little effect on the germination of Amorpha fruticosa L. seeds, suggesting that the species possesses a certain degree of drought tolerance [6]. At the same time, Amorpha fruticosa L. is a shrub species with strong tolerance to saline-alkali stress conditions, it can grow normally in soil with a salinity of 100 mM and can also significantly reduce soil salinization [7]. Jiang Yuanfang and colleagues found through their research that, after 35 days of stress exposure to 150 mmol/L NaHCO₃, two-year-old Amorpha fruticosa L. seedlings showed no significant changes in either morphology or physiological indicators. The external morphology of the plants most clearly reflects their stress-resistant growth status [8].

Ethylene Response Factor(AP2/ERF) are a large family of transcription factors found primarily in plants, characterized by the presence of an AP2/ERF domain involved in DNA binding. This domain is approximately 60–70 amino acids in length and consists of a three-stranded β-sheet and an α-helix that runs nearly parallel to the sheet, arginine and tryptophan are the key residues in the β-sheet responsible for DNA binding [9]. AP2/ERFs are widely distributed throughout the plant kingdom and play a critical role in the transcriptional regulation of plant growth and development as well as in defense responses [10]. AP2/ERF is divided into five subfamilies: AP2 (APETALA2), ethylene response factors (ERF), drought response element-binding proteins (DREB), RAV, and Soloist [11]. AfRAP2 possesses an AP2 domain and belongs to the DREB subfamily of the AP2/ERF transcription factor family.

Dehydration-responsive element-binding proteins (DREBs) are the most widely distributed AP2/ERF transcription factor subfamily involved in abiotic stress responses [12]. The DREB can specifically bind to DRE/CRT (G/ACCGAC) cis-elements to cope with abiotic stress. These proteins typically contain a single AP2 domain of 60–70 amino acids, comprising two conserved blocks, YRG and RAYD elements. The main function of YRG element is to bind to the cis-acting elements of target genes. The amino acids V14 and E19 within AP2 domain are crucial for this binding, with V14 being particularly critical for DNA-binding specificity. RAYD element mainly regulated the binding of AP2 conserved domains to cis-acting elements through structural conformation or protein–protein interactions [13]. The introduction of DREBs can enhance a plant’s tolerance to abiotic stresses, for example, DREB1A driven by the rd29A promoter results in increased drought tolerance in wheat plants [14], Overexpression of DREB1A in rice plants leads to improved tolerance to drought and salinity [15], wheat plants transformed with GhDREB from cotton accumulate higher levels of soluble sugars and chlorophyll in their leaves following stress treatment, resulting in improved tolerance to drought, high salinity, and freezing stress [16]; The genetically modified banana MusaNAC042 exhibits tolerance to salt and drought stress, while also regulating the transcriptional levels of various CBF/DREB proteins in the genetically modified banana [17]. The AP2 domain was first identified in the Arabidopsis AP2 protein, which plays a role in flower development [18]. AP2/ERF transcription factors can form cross-regulatory networks in conjunction with hormone signaling, thereby enhancing plant responses to and tolerance of abiotic stresses [19]. For example, in sweet potato, IbRAP4.2 promotes lateral root formation and exhibits drought resistance [20]. in Arabidopsis, AtRAP2.11 participates in the low-potassium signaling pathway [21]. The strong expression of ERF3-like genes in transgenic banana plants suggests that MusaNAC29-like transcription factors may play a role in enhancing tolerance to abiotic stress [22]. Based on our laboratory’s transcriptomic analysis of Amorpha fruticosa L., this gene is upregulated under drought and alkaline-salinity stress. However, to date, there have been no reports on the stress-responsive functions of the AfRAP2 gene.

Based on this, this study uses Amorpha fruticosa L. as the research subject. Gene and protein information for AfRAP2 was retrieved from NCBI, the LOCUS ID is MT13552, and the protein name is QQL14324.1. Bioinformatics analysis revealed that AfRAP2 contains an AP2 domain, which belongs to the DREB subfamily of the AP2/ERF transcription factor family. According to phylogenetic tree analysis, AfRAP2 is closely related to LaEREBP from white lupine. To investigate the role of the AfRAP2 gene in yeast resistance, we performed spatial-specific expression of the AfRAP2 gene and assessed its response to abiotic stresses such as NaHCO₃ and PEG6000. Using subcellular localization and Agrobacterium-mediated genetic transformation techniques, we overexpressed the AfRAP2 gene in tobacco and Populus davidiana×P. alba var. pyramidalis. Through phenotypic observations and data analysis, we elucidated the stress resistance mechanisms of the AfRAP2 gene.

Materials

Amorpha fruticosa L. seeds were collected from our laboratory’s Ande field trial site, and we have obtained the necessary collection permit from our laboratory. Fresh Amorpha fruticosa L. leaves at the three-leaf stage were collected, flash-frozen in liquid nitrogen, ground into powder, and used to extract total RNA, which was then reverse-transcribed into cDNA. Tissue-cultured seedlings of Populus davidiana×P. alba var. pyramidalis were maintained in-house by our laboratory.

Methods

Bioinformatics analysis

The ORF and amino acid sequence of AfRAP2 were retrieved from the online database NCBI. Online software was used to predict the protein’s primary structure (ProtScale), secondary structure (Prabi), and tertiary structure (SWISS-MODEL). Phosphorylation sites in the AfRAP2 gene were predicted using NetPhos 3.1. A phylogenetic tree was constructed using MEGA 11 software. To investigate the function of the AfRAP2 promoter and identify its specific expression sites, the first 314 bp of AfRAP2 were sequenced, and the promoter elements within this region were analyzed using Plant CARE.

Promoter activity, spatiotemporal expression, and subcellular localization of AfRAP2

Based on the obtained AfRAP2 ORF sequence, specific primers were designed using Primer5 to obtain the CDS sequence of the AfRAP2 gene. To investigate the expression patterns of the AfRAP2 gene in different organs of Amorpha fruticosa L., tissues including roots, stems, leaves, flowers, catkins, and bark were collected. Additionally, four-week-old Amorpha fruticosa L. seedlings were obtained using hydroponic culture. Total RNA was extracted from the seven tissues— roots, stems, leaves, flowers, spica, skin, and seedlings—using the Trizol method [23] and reverse-transcribed into cDNA. To investigate changes in the expression levels of the AfRAP2 gene in Amorpha fruticosa L. under stress conditions, Amorpha fruticosa L. seedlings were grown hydroponically until they reached four weeks of age. Seedlings with consistent growth were then selected and subjected to time-gradient stress treatments using 50 mM NaHCO₃, 15% PEG 6000. Total RNA was extracted and reverse-transcribed into cDNA. Using cDNA from different organs of Amorpha fruticosa L. and cDNA from seedlings subjected to 50 mM NaHCO₃ and 15% PEG 6000, RT-qPCR was performed using the MxPro-Mx3000P [24] system. Relative gene expression levels were calculated using the 2^(-ΔΔCt) method, and the data were analyzed using GraphPad software.

Analysis of the first 314 bp of the promoter using Plant CARE. The constructed pGWB3-AfRAP2pro-Gus vector was validated using a specific primer (Table S1). The validated pGWB3-AfRAP2pro-Gus vector was then transformed into Agrobacterium, which was used to infect fresh young leaves of 4-week-old wild-type tobacco. DNA was extracted from transgenic tobacco seedlings using the CTAB method [25]. Using the extracted DNA as a template and the AfRAP2-specific primer (Table S1), PCR was performed. The plasmid served as a positive control and the wild-type as a negative control to determine whether a single target band was obtained. Wild-type tobacco seeds and pGWB3-AfRAP2pro-Gus T1-generation tobacco seeds were spotted onto 1/2 MS medium and medium containing Hyg (50 mg/L) for resistance screening. Transgenic plant tissues were stained using GUS staining solution, incubated in the dark at 37 °C for 2 days, and then decolorized with alcohol.

Using Primer5 software, we designed primers specific for pEH12-AfRAP2-GFP to construct the pEH12-AfRAP2-GFP vector, which was then verified via PCR and restriction enzyme digestion. The plasmid from the verified vector was transformed into Agrobacterium. The Agrobacterium culture, confirmed to be correct, was injected into 2-month-old tobacco leaves using the tobacco leaf injection method for subcellular localization by fluorescence microscopy. On the day of observation, the injected tobacco leaves were soaked in DAPI dye [26] and then rinsed with PBS.

Analysis of AfRAP2 gene expression and stress tolerance in yeast

To verify the expression and stress resistance of the AfRAP2 gene in yeast, a recombinant plasmid, pYES2-AfRAP2, was constructed using T4 DNA ligase. The verified pYES2-AfRAP2 plasmid was then transformed into the yeast strain INVSc1, and the strain pYES2-AfRAP2-INVSc1, which successfully harbored the recombinant plasmid, was selected using a specific primer (Table S1).

The successfully identified pYES2-AfRAP2-INVSc1 yeast strain and the pYES2-INVSc1 yeast strain transformed with the pYES2 empty vector were cultured in YPD liquid medium until the OD₆₀₀ reached approximately 0.6. The yeast cell suspensions were then diluted with saline to five concentration gradients: 10⁻¹, 10⁻², 10⁻³, 10⁻⁴, and 10⁻⁵ [27]. For each concentration, 3 µL of bacterial suspension was spotted onto YPG solid medium containing mannitol (1 mL/L, 2 mL/L, 3 mL/L), sorbitol (1 mL/L, 2 mL/L, 3 mL/L), or NaHCO₃(10 mM/L, 20 mM/L, 30 mM/L) as stress conditions, with blank YPG solid medium serving as the control. The plates were incubated at 30 °C for 2–3 days, after which differences in yeast colony growth were recorded.

Genetic transformation and resistance analysis of AfRAP2-overexpressing tobacco

Transgenic lines were obtained by infecting four-leaf-stage tobacco plants with an Agrobacterium suspension containing pEH12-AfRAP2-GFP via leaf infection. DNA was extracted from the transgenic tobacco plants and analyzed via PCR using AfRAP2-specific primers, with the plasmid serving as a positive control and the wild-type (WT) as a negative control. Correctly identified T3-generation transgenic tobacco plants and wild-type tobacco plants were grown to the four-leaf stage, and relative expression levels were determined using quantitative real-time PCR (RT-qPCR).

To analyze tobacco resistance during the germination stage, wild-type tobacco seeds and transgenic AfRAP2 tobacco seeds T3-#1, T3-#3, and T3-#5 were sown at 20 seeds each on 1/2 MS medium and 1/2 MS medium supplemented with NaHCO₃ (2 mM, 3 mM, 5 mM) and sorbitol (250 mM, 275 mM, 300 mM). The number of germinated seeds was recorded starting from day 2, data were collected through day 10, and the daily germination rate was calculated, phenotypic observations were documented and documented by photography on day 10.

To analyze tobacco resistance during the seedling stage, wild-type T3-generation tobacco seeds and transgenic AfRAP2 T3-generation tobacco seeds (T3-#1, T3-#3, and T3-#5) collected from the same batch were sown on 1/2 MS medium and grown vertically for 10 days. Subsequently, seedlings with consistent growth were selected and placed on either 1/2 MS medium or 1/2 MS medium supplemented with 2 mM NaHCO₃,4 mM NaHCO₃,6 mM NaHCO₃. Observations were made and photographs taken daily.

To analyze tobacco resistance during the seedling stage, wild-type tobacco seeds and transgenic AfRAP2 tobacco seeds (T3-#1, T3-#3, and T3-#5) collected from the same batch were sown in soil. Once the seedlings reached the same growth stage, they were subjected to a 0.5 M NaHCO₃ root drench treatment with 30 mL of 0.5 M NaHCO₃ solution was applied every two days. Photographs were taken on days 3, 7, 10, and 14 to record stress phenotypes. Leaves were collected from the same position on each plant to measure physiological parameters, chlorophyll fluorescence [28], and relative expression levels. Concurrently, natural drought stress was applied for 7 and 14 days, with photographs taken for record-keeping. Leaves from days 0, 3, 7, 10, and 14 of the treatment were ground, scraped into powder, and mixed with 2 mL of PBS (pH = 7.8) to prepare enzyme solutions. Malondialdehyde (MDA) [29], catalase (CAT) [30], superoxide dismutase (SOD) [31], and peroxidase (POD) [32] in wild-type and transgenic plants were measured to evaluate oxidative stress and antioxidant enzyme activities.

Genetic transformation and resistance analysis of AfRAP2-overexpressing Populus davidiana×P. alba var. pyramidalis

A suspension of Agrobacterium containing pEH12-AfRAP2 was used to infect fresh, tender leaves of 4-week-old wild-type Populus davidiana×P. alba var. pyramidalis. DNA was extracted from the transgenic Populus davidiana×P. alba var. pyramidalis and identified using a specific primer (Table S1).

Wild-type (WT), the correctly identified #2 transgenic Populus davidiana×P. alba var. pyramidalis plant, transgenic Populus davidiana×P. alba var. pyramidalis plants #3 and #4, and punch 1.4 cm diameter holes in the third, fourth, fifth, and sixth leaves from the apical bud. Immerse the punched leaves in solutions containing 0 mM, 200 mM, 250 mM, and 300 mM NaHCO₃, and observe the phenotypes after 2 days.

Wild-type Populus davidiana×P. alba var. pyramidalis plants and AfRAP2-overexpressing lines #2, #3, and #4 with consistent growth were selected. Stem segments containing the apical buds with 8 leaves were cut and transferred to 1/2 MS medium supplemented with 5 mM NaHCO₃ and 200 mM sorbitol. 1/2 MS medium served as the control. Phenotypes were observed after 7 days. The expression levels of PdbCAT, PdbPOD, PdbSOD, and PdbSDS genes were determined by quantitative real-time PCR (qPCR) in wild-type plants and AfRAP2-overexpressing lines of Populus davidiana×P. alba var. pyramidalis plantsis under conditions of 5mM NaHCO₃and 200 mM sorbitol.

Statistics analysis

Statistical analyses were performed using SPSS (v26.0). For comparisons involving two groups, unpaired two-tailed Student's t-tests were used. For multi-group comparisons (three or more groups), the error bars represent the standard errors of three biological replicates, with significant differences determined at the p < 0.05 level. Lowercase letters (a, b, c, etc.) denote statistically significant differences (p < 0.05). Experiments were replicated at least three times, and representative data are shown. Bar charts and box plots were generated using GraphPad Prism 8 and Origin 2025b, and figures were assembled in Adobe Illustrator CS6.

Results and analysis

Bioinformatics analysis

To analyze the biological characteristics of the AfRAP2 gene, we searched for AfRAP2 gene information on NCBI. The LOCUS ID is MT13552, the protein name is QQL14324.1, the corresponding UniProt protein ID is A0A7T7DN92, the full-length CDS is 513 bp, and it encodes 170 amino acids. The online prediction of the molecular formula is C₈₂₈H₁₃₂₅N₂₅₃O₂₆₄S₆, with a molecular weight of 19,24 kDa.

The primary structure of the AfRAP2 protein from Amorpha fruticosa L. contains an AP2 domain, with valine and glutamic acid at amino acid positions 14 and 19, respectively (Fig. 1A). This protein belongs to the DREB subfamily within the AP2/ERF transcription factor family and features a secondary structure consisting of four components: 23.53% α-helix, 16.47% extended strand, 3.53% β-turn, and 56.47% random coil (Fig. 1B). SWISS-MODEL was used to predict the protein’s tertiary structure online and construct a three-dimensional protein model (Fig. 1C).

Fig. 1.

Bioinformatics analysis of the protein encoded by AfRAP2. A The primary structure of the protein, including the AP2 domain, B Secondary structure prediction, including linear alignment and curve distribution, C Tertiary structure prediction of the protein, D Phosphorylation site prediction. E Construction of a phylogenetic tree, with ● indicating the branch occupied by AfRAP2, Note: La(Lupinus albus), An(Ammopiptanthus nanus), Am(Ammopiptanthus mongolicus), Ap(Abrus precatorius), Gm(Glycine max), Ss(Spatholobus suberectus), Va(Vigna angularis), Mf(Medicago falcataL), Pa(Populus alba), Cc(Cajanus cajan), Sm(Solanum melongena L). ID:LaEREBP(ID:101512899), AnCBF1(ID:89984318 ), AmDREB(100499747), AiERF011(ID:107648576) ApRAP2-1(ID: 841117), GmRAP2-1(ID: 100801528), GmDREB(ID: 100499747), SsRAP2-1(ID: 117849116), VaRAP2-1(ID: 108341845), MpRAP2-1(ID: 11432890), HhDREB(ID: 110911103), TpRAP2-1(ID: 18591157), MfERF014(ID: 136540907), MtRAP2-1(ID: 11432890), PaRAP2-1-Like(ID: 133926311), ApRAP2-1-Like(ID: 127264295), SsRAP2-1.1(ID: 125971988), CcRAP2-1(ID: 109818492), SmDREB2(ID: 110429986), F. AfRAP2 promoter elements

Phosphorylation site prediction of the AfRAP2 protein using NetPhos 3.1 identified a total of 22 amino acid phosphorylation sites with scores exceeding the 0.5 threshold in the amino acid sequence (Fig. 1D). These consisted primarily of 14 serine (Ser) residues, along with 7 threonine (Thr) residues and 1 tyrosine (Tyr) residue. By constructing a phylogenetic tree of AfRAP2, it was found that AfRAP2 is closely related to LaEREBP from Lathyrus albus (Fig. 1E). Analysis of the first 314 bp of the promoter using Plant CARE revealed the presence of expression elements such as ABRE, MYB, and G-box (Fig. 1F), along with their specific binding sites and functions (Table S2).

Promoter activity, spatiotemporal expression, and subcellular localization of AfRAP2

The recombinant plasmid pGWB3-AfRAP2pro-GUS was transformed into tobacco, and agarose gel electrophoresis was used to verify its transformation into the tobacco plants (Fig. S1). Real-time quantitative PCR (qPCR) was employed to analyze the expression levels in the transgenic tobacco plants. The results showed that expression levels were elevated in all transgenic lines, with lines #1, #2, and #3 exhibiting the highest expression levels (Fig. 2A). These three lines were selected for subsequent experiments. This also confirmed that the AfRAP2pro gene has been integrated into the tobacco genomic DNA. At the same time, wild-type tobacco seeds and T1-generation tobacco seeds were sown on 1/2 MS medium and on medium containing Hyg (50 mg/L) for resistance screening (Fig. S2). Transgenic tobacco plants capable of growing on 1/2 MS medium containing Hygromycin were transplanted into soil. GUS histochemical staining of seedlings and flowers was subsequently performed, and blue staining was predominantly detected in the anthers and stigmas (Fig. 2B).

Fig. 2.

Analysis of the AfRAP2 promoter, tissue, organ, and temporal specificity, and subcellular localization. A RT-qPCRaAnalysis of transgenic tobacco for promoter identification, B Histochemical staining of the anthers and stigmas of transgenic tobacco, C RT-PCR analysis of AfRAP2 gene expression in Amorpha fruticosa L. tissues and organs, D-E Expression profiles of the AfRAP2 gene in response to 15% PEG and NaHCO₃ treatments, F Subcellular localization of AfRAP2. Note: The error bars represent the standard errors of three biological replicates, with significant differences determined at the p < 0.05 level. Lowercase letters (a, b, c, etc.) denote statistically significant differences (p < 0.05)

Real-time quantitative PCR (RT-qPCR) analysis revealed that the AfRAP2 gene is expressed in all tissues and organs of Amorpha fruticosa L. (Fig. 2C). During the growth of Amorpha fruticosa L., the expression levels of the AfRAP2 gene vary among roots, stems, leaves, flowers, spica, skin, and seedlings. The highest expression was observed in leaves, followed by spica and seedlings, and lowest in stems, with leaf levels approximately 80-fold higher than those in stems. Under PEG6000 stress, expression in both roots and leaves was significantly higher than those in the control group at 12 , 24 , and 48 h, reaching a maximum at 48 h. Specifically, expression in the roots was 3.6-fold that of the control group, while expression in the leaves was 3.2-fold that of the control group (Fig. 2D). Under NaHCO₃ stress, expression levels in Amorpha fruticosa L. roots reached approximately 2.7-foid those of the control at 6, 12, 24, and 48 h, while expression levels in Amorpha fruticosa L. leaves reached approximately 2–3-fold increases at the same time points (Fig. 2E). This suggests that the AfRAP2 gene may respond to changes in saline-alkali stress and drought.

Different protein functions result in different subcellular localizations. The constructed pEH12-AfRAP2-GFP recombinant expression vector was transformed into Agrobacterium. After transiently transfecting tobacco leaf epidermal cells, the distribution of the GFP green fluorescent signal was observed under a fluorescence microscope, revealing that the fluorescent signal was primarily localized in the cell nucleus. The control group exhibited fluorescence signals distributed from the cytoplasm to the nucleus, whereas the fluorescence signals of the AfRAP2::GFP fusion protein were primarily localized in the nucleus(Fig. 2F). Thus, it can be concluded that the AfRAP2 protein is primarily localized in the nucleus.

Analysis of AfRAP2 gene expression and stress tolerance in yeast

The recombinant pYES2-AfRAP2 plasmid and empty pYES2 plasmids were transformed into the INVSc1 strain and diluted to five concentrations: 10⁻¹, 10⁻², 10⁻³, 10⁻⁴, and 10⁻⁵. For each concentration gradient, 3 µL of the yeast suspension was spotted onto YPG solid medium containing different concentrations of mannitol, sorbitol, and NaHCO₃. After the medium had dried, the plates were sealed. Yeast cells transformed with the empty pYES2 vector served as the control. The plates were incubated at 30 °C for 3 days, and differences in yeast growth were recorded (Fig. 3). On YPG solid medium without treatment, there was no difference in growth between the control and experimental groups. However, on YPG solid medium supplemented with increasing concentrations of mannitol and sorbitol, the experimental group exhibited superior growth compared to the control, indicating that the yeast expressing the pYES2-AfRAP2 gene possesses a certain degree of drought tolerance. Similarly, when grown on YPG solid medium containing different concentrations of NaHCO₃, the control group exhibited weaker growth than the experimental group as the concentration increased, suggesting that the yeast in the experimental group possesses a certain degree of tolerance to alkaline salt. Collectively, these results demonstrate that yeast expressing the pYES2-AfRAP2 gene exhibits both drought resistance and alkaline salt tolerance.

Fig. 3.

Analysis of the stress resistance of the AfRAP2 gene under mannitol, sorbitol, and NaHCO₃ stress

Genetic transformation and resistance analysis of AfRAP2-overexpressing tobacco

Schematic diagram of the pEH12 vector T-DNA construction (Fig. 4A). Transgenic tobacco plants were obtained through Agrobacterium-mediated transformation using the successfully cloned plant expression vector pEH12-AfRAP2. To determine whether tobacco lines expressing the pEH12-AfRAP2 gene had been successfully generated, DNA was extracted from both wild-type and transgenic tobacco plants. PCR analysis was performed, with wild-type tobacco serving as the negative control(Fig. S3). Bands of the same size as the positive control appeared in the DNA of all six transgenic tobacco plants, whereas no bands were observed in the negative control, confriming that AfRAP2 had integrated into the tobacco genome.

Fig. 4.

Analysis of AfRAP2 transgenic tobacco resistance during the germination and seedling stages. A Schematic diagram of the T-DNA structure of the pEH12-AfRAP2 vector, B Transcription levels of the AfRAP2 gene in transformed tobacco, C Germination of transgenic tobacco on 1/2 MS, 1/2 MS + 2 mM NaHCO₃, and 1/2 MS + 250 mM sorbitol. Wild-type (WT), transgenic lines (OE1, OE3, OE5), D–F Germination rates under different stress conditions, G Seedling-stage stress of transgenic tobacco on NaHCO₃. Wild-type (WT), transgenic lines (OE1, OE3, OE5), H–I Fresh weight and chlorophyll content under seedling-stage NaHCO₃ stress. Note: The error bars represent the standard errors of three biological replicates, with significant differences determined at the p < 0.05 level. Lowercase letters (a, b, c, etc.) denote statistically significant differences (p < 0.05)

RNA was extracted from the transgenic plants identified as positive, reverse-transcribed into cDNA, and analyzed by RT-qPCR (Fig. 4B). The expression levels in the transgenic plants were significantly higher than those in the wild-type. Among them, plants #1, #3, and #5 exhibited higher expression levels than the overexpressing plants. These three lines were subsequently selected for further resistance analysis.

To study the function of the AfRAP2 gene, wild-type tobacco seeds and transgenic tobacco seeds T3-#1, T3-#3, and T3-#5 were co-sown on 1/2 MS, 1/2 MS + 2 mM NaHCO₃, and 1/2 MS + 250 mM sorbitol media (Fig. 4C).1/2 MS + 3 mM NaHCO₃, 1/2 MS + 5 mM NaHCO₃, 1/2 MS + 275 mM sorbitol, 1/2 MS + 300 mM sorbitol (Fig. S4). Germination was recorded daily from day 2 to day 10, and germination rates were calculated. Phenotypes were photographed on day 10. (Fig. 4D-F). On 1/2 MS medium, wild-type and transgenic tobacco exhibited similar growth. Under NaHCO₃ stress treatment, transgenic tobacco grew better than the wild-type, although the number of germinating seeds gradually decreased with increasing concentrations. Under non-stress conditions, there was no significant difference in germination rates between wild-type and transgenic tobacco, however, under 2 mM NaHCO₃ stress, the germination rate of wild-type tobacco was lower than that of transgenic tobacco starting from day 3. Under sorbitol stress treatment, wild-type tobacco barely germinated, whereas transgenic tobacco maintained the ability to germinate. In the absence of stress, there was no significant difference in germination rates between the two groups, but under sorbitol stress, the germination rate of transgenic tobacco was significantly higher than that of the wild-type. This suggests that AfRAP2-transgenic tobacco exhibits NaHCO₃ and drought tolerance during the germination stage.

To further investigate whether NaHCO₃ tolerance persists during the seedling stage, wild-type and transgenic seeds were germinated for 7 days on 1/2 MS medium and 1/2 MS medium containing 50 mg/L hygromycin, after which they were transferred to 1/2 MS, 1/2 MS + 2 mM NaHCO₃, 1/2 MS + 4 mM NaHCO₃, and 1/2 MS + 6 mM NaHCO₃ media for vertical cultivation for 3 days. Photographs were taken (Fig. 4G), and fresh weight and chlorophyll content were subsequently measured (Fig. 4H–I). There were no significant differences between wild-type and transgenic plants on 1/2 MS medium. As the stress concentration increased, the leaves of wild-type tobacco gradually turned yellow and, notably, turned white under 6 mM NaHCO₃ stress, whereas the transgenic tobacco remained green. Subsequently, statistical analyses were performed on the fresh weight and chlorophyll content of the plants after stress. With increasing NaHCO₃ stress concentration, the fresh weight and chlorophyll content were significantly higher in transgenic tobacco plants than in wild-type plants. These results indicate that AfRAP2 transgenic tobacco possess NaHCO₃ tolerance during the seedling stage.

To further investigate the function of AfRAP2, in this study, T3-generation tobacco seedlings were treated with 30 mL of 500 mM NaHCO₃ via root drenching every two days during the seedling stage(Fig. 5A). Wilting was first observed at day 3 post-treatment, followed by progressive leaf chlorosis and wilting from day 7. By day 14, the leaves of the wild-type plants had completely turned yellow and the plants had died, while the transgenic plants exhibited only slight leaf yellowing and survived. Simultaneously, chlorophyll fluorescence spectra were measured for leaves of wild-type and transgenic plants before treatment and 14 days after treatment (Fig. 5B). Before treatment, there was no difference in the maximum photochemical absorption (Fv/Fm) of photosystem II in the chloroplasts of the leaves, 14 days after treatment, the maximum absorption in the transgenic leaves was significantly greater than that in the wild-type leaves. The results indicate that under saline-alkali stress, transgenic AfRAP2 tobacco plants suffered less damage and exhibited a certain degree of tolerance to saline-alkali stress.

Fig. 5.

Tolerance of AfRAP2 transgenic tobacco to NaHCO₃ and drought stress during the vegetative stage. A T3-generation tobacco plants treated with 500 mM NaHCO₃ via root drenching. Wild-type (WT), transgenic lines (OE1, OE3, OE5), B Chlorophyll fluorescence(Fv/Fm) measurements in leaves of wild-type and transgenic plants before treatment and 14 days after treatment, C Levels of malondialdehyde (MDA) and activities of catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD) under NaHCO₃ stress, D Natural drought treatment applied to T3-generation tobacco seedlings during the seedling stage. Wild-type (WT), transgenic lines (OE1, OE3, OE5), E Levels of malondialdehyde (MDA), catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD) under drought stress

Enzyme extracts were prepared from leaves at 0, 3, 7, 10, and 14 days after treatment, and the levels of malondialdehyde (MDA), catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD) were measured in wild-type and transgenic plants(Fig. 5C). As the duration of stress increased, the levels of all four parameters continued to rise. MDA content were higher in wild-type plants than in transgenic plants, while the activities of CAT, SOD, and POD were higher in transgenic lines than in wild-type lines. At 14 days post-treatment, WT plants had died, and reliable enzyme activity measurements could not be obtained from the dead tissues. It is therefore inferred that as the duration of stress increases, tobacco plants are continuously subjected to stress damage, and transgenic plants toletate adverse environmental conditions by producing CAT, SOD, and POD to scavenge free radicals within the plant.

To investigate whether transgenic AfRAP2 tobacco plants also exhibit drought tolerance during the seedling stage, T3-generation tobacco seedlings were subjected to natural drought treatment for 7 and 14 days(Fig. 5D). Before subjecting the plants to natural drought conditions, seedlings of uniform growth were selected. On the 7th day of natural drought, wild-type tobacco leaves exhibited wiltingand drooping, while the transgenic tobacco plants remained in good condition, on the 14th day of natural drought, wild-type tobacco plants showed wilting and dry leaves, whereas the transgenic tobacco plants survived with only slightly wilted leaves. Enzyme extracts were prepared from leaves at 0, 7, and 14 days of natural drought treatment, and the levels of MDA, CAT, SOD, and POD were measured in both wild-type and transgenic plants(Fig. 5E). As the duration of drought increased, the levels of MDA, CAT, SOD, and POD continued to rise. The MDA content in wild-type plants was significantly higher than that in transgenic plants, however, MDA levels dropped rapidly after 14 days of drought stress, while the levels of CAT, SOD, and POD in the transgenic lines were significantly higher than those in the wild-type lines. These results indicate that as the duration of stress increases, tobacco plants are continuously subjected to stress damage, transgenic plants tolerate adverse environmental conditions by producing CAT, SOD, and POD to scavenge free radicals within the plant.

Genetic transformation and resistance analysis of AfRAP2-overexpressing Populus davidiana×P. alba var. pyramidalis

To verify whether the Populus davidiana×P. alba var. pyramidalis line had been successfully transformed with the pEH12-AfRAP2 gene, DNA was extracted from both wild-type Populus davidiana×P. alba var. pyramidalis and transgenic Populus davidiana×P. alba var. pyramidalis. PCR analysis was performed, using wild-type Populus davidiana×P. alba var. pyramidalis as a negative control and the pEH12-AfRAP2 recombinant plasmid as a positive control (Fig. S5). RNA was extracted from selected positive transgenic Populus davidiana×P. alba var. pyramidalis plants, reverse-transcribed into cDNA, and analyzed by RT-qPCR. The expression levels in the transgenic plants were significantly higher than those in the wild-type. Among them, plants #2, #3, and #4 exhibited highest expression levels than the other lines(Fig. 6A).

Fig. 6.

Tolerance of AfRAP2 transgenic Populus davidiana×P. alba var. pyramidalis to NaHCO₃ and drought stress during the vegetative stage. A Relative expression levels of the AfRAP2 gene in transgenic Populus davidiana×P. alba var. pyramidalis, B Punch-hole leaf stress in wild-type (WT) and transgenic Populus davidiana×P. alba var. pyramidalis lines. Wild-type (WT), transgenic lines (OE2, OE3, andOE4), C Analysis of saline-alkali stress and drought tolerance in AfRAP2-transgenic Populus davidiana×P. alba var. Pyramidalis. D RT‑qPCR analysis was performed on related enzyme genes in wild‑type (WT) and transgenic Populus davidiana×P. alba var. pyramidalis under NaHCO₃ and drought stress. Wild-type (WT), transgenic lines (OE2, OE3, OE4). Note: The error bars represent the standard errors of three biological replicates, with significant differences determined at the p < 0.05 level. Lowercase letters (a, b, c, etc.) denote statistically significant differences (p < 0.05)

To determine whether the AfRAP2 gene confers NaHCO₃ tolerance in woody plants similar to that observed in tobacco, perforated leaves from wild-type (WT) and transgenic Populus davidiana×P. alba var. pyramidalis lines were subjected to stress treatment (Fig. 6B). Prior to stress treatment, all leaf discs were in good condition and exhibited uniform color. Two days after saline-alkali stress, the control group retained uniform color. Under 200 mM NaHCO₃ stress, the outer ring of the wild-type Populus davidiana×P. alba var. pyramidalis perforated leaves darkened, indicating damage, whereas the transgenic Populus davidiana×P. alba var. pyramidalis remained intact. Under 250 mM NaHCO₃ stress, more than half of the area of the wild-type Populus davidiana×P. alba var. pyramidalis perforated leaves was damaged. Under 300 mM NaHCO₃ stress, the wild-type Populus davidiana×P. alba var. pyramidalis perforated leaves had turned completely black, indicating severe damage, whereas the transgenic Populus davidiana×P. alba var. pyramidalis perforated leaves remained in good condition. These results indicate that the AfRAP2 gene confers a certain degree of NaHCO₃ tolerance to the transgenic Populus davidiana×P. alba var. pyramidalis.

To determine whether the AfRAP2 gene confers alkali and drought tolerance in Populus davidiana×P. alba var. pyramidalis, wild-type and three AfRAP2-overexpressing transgenic lines #2, #3 and #4 with comparable growth rates were selected. Stem segments from the apical buds, each bearing 8 leaves, were transferred to 1/2 MS medium supplemented with 5 mM NaHCO₃ or 200 mM sorbitol, with 1/2 MS medium alone serving as the control. Phenotypes were observed after 7 days (Fig. 6C). Under control conditions, both the wild-type and transgenic lines grew normally. Under 5 mM NaHCO₃ stress, the wild-type plants were severely affected, with curled and yellowing leaves, although the transgenic lines were also affected by NaHCO₃ stress, the degree of leaf curling was milder compared to the WT plants. Under 200 mM sorbitol stress, both WT and transgenic lines survived, but WT plants showed slight wilting, whereas transgenic lines remained turgid. The experimental results indicate that the AfRAP2-overexpressing Populus davidiana×P. alba var. pyramidalis line possesses a certain degree of tolerance to NaHCO₃ salts and drought stress. To investigate the molecular mechanisms underlying the NaHCO₃ tolerance and drought resistance of AfRAP2-transgenic plants, the expression levels of PdbCAT, PdbSOD, PdbPOD, and PdbSOS were also measured by RT-qPCR in both wild-type and transgenic plants following NaHCO₃ and sorbitol stress (Fig. 6D). Prior to treatment, no significant differences were observed in the expression of PdbCAT, PdbSOD, PdbPOD, and PdbSOS between wild-type and transgenic plants. After treatment, the expression levels of these four genes were found to be significantly elevated in transgenic lines compared with those in wild-type plants. From these results, it can be inferred that AfRAP2 positively regulates the expression of these enzyme-encoding genes under NaHCO₃ and drought stress, and that by activating their expression, it enhances the NaHCO₃ tolerance and drought resistance of Populus davidiana×P. alba var. pyramidalis.

Discussion

Amorpha fruticosa L. is a widely distributed and highly adaptable legume capable of growing in poor, arid, and slightly saline-alkali soils. During its growth, the plant is subjected to various abiotic stresses, such as drought and salinity [33]. When plants face such stresses, AP2/ERF transcription factors—either acting independently or binding to the promoters of target genes—induce downstream response genes. This helps plants withstand abiotic stress by producing endogenous substances such as malondialdehyde (MDA), catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD), or by altering their own morphology [34]. In our previous research, the differentially expressed gene AfRAP2 was identified through transcriptomic sequencing of Amorpha fruticosa L. under drought-simulated conditions using PEG6000. In this study, the AfRAP2 gene was heterologously expressed in Nicotiana benthamiana and Populus davidiana×P. alba var. pyramidalis. After subjecting the transgenic plants to simulated drought and saline-alkali stress treatments, the results revealed that AfRAP2 enhanced the drought tolerance to saline-alkali stress conditions in the transgenic plants (Figs. 4, 5 and 6).

Our study found that AfRAP2 contains an AP2 domain, with valine at position 14 and glutamic acid at position 19, placing it within the DREB subfamily. DREB serves as a core transcriptional regulator of abiotic stress responses [35]. Overexpression of DREB genes significantly enhances the transgenic plants’ tolerance to diverse abiotic stresses, such as drought, salinity, and low temperature [36]. Analysis of spatial expression specificity in Amorpha fruticosa L. plants revealed expression in roots, stems, leaves, flowers, pods, bark, and seedlings, with the highest expression levels observed in leaves. In Arabidopsis, the RAP2.6 gene is expressed in seedlings, stems, and rosette leaves, with the highest expression levels observed during the seedling stage [37]. Online prediction tools indicated that the AfRAP2 protein localizes to the cytoplasm or nucleus. To verify the subcellular localization of AfRAP2, we performed Agrobacterium-mediated transformation in tobacco and found that the AfRAP2 protein is predominantly localized to the nucleus. To verify whether the 5’-end fragment of the AfRAP2 gene possesses promoter activity, the 314-bp fragment upstream of the AfRAP2 start codon was cloned, and transgenic plants were generated. The entire seedlings and flowers were stained, and the appearance of blue coloration in the anthers and stigmas indicated that AfRAP2 was expressed in these tissues. To verify whether AfRAP2 confers tolerance in yeast, we introduced the target gene into yeast for resistance experiments and treated the cells with sorbitol, mannitol, and NaHCO₃. We found that the transgenic yeast grew better than the control group (Fig. 3). Subsequently, we obtained tobacco plants transgenic for the AfRAP2 gene and harvested T3-generation homozygous seeds. Germination experiments were conducted on T3 seeds under NaHCO₃ and sorbitol stress, revealing that the germination rate of transgenic tobacco seeds was significantly higher than that of the wild-type under stress conditions. Additionally, tobacco seedlings were cultured vertically under NaHCO₃ stress for 3 days. The wild-type tobacco turned white, while the transgenic tobacco remained green. Measurements of fresh weight and chlorophyll content revealed that the transgenic tobacco performed significantly better than the wild-type, indicating that transgenic tobacco possesses stronger tolerance to NaHCO₃ During the seedling stage, NaHCO₃ root drenching was performed. Observations of the phenotype and results from chlorophyll fluorescence imaging after treatment showed that the wild-type tobacco suffered greater damage, while the transgenic tobacco exhibited better growth than the wild-type. Measurements of MDA, SOD, POD, and CAT levels also indicated that the transgenic tobacco outperformed the wild-type. Similarly, under salt stress conditions, Arabidopsis plants overexpressing MbERF12 exhibited increased POD, CAT, and SOD activities, as well as reduced MDA levels [38]; in contrast, transgenic Arabidopsis plants overexpressing ScDREB5 had lower levels of MDA and hydrogen peroxide (H₂O₂), while SOD, POD, and CAT activities were elevated [39]. Under natural drought stress, the results were consistent with those observed under NaHCO₃ treatment. Notably, the MDA content in wild-type plants decreased sharply after 14 days of drought stress, which was presumably due to the death of the seedlings caused by the prolonged drought period. These findings indicate that the AfRAP2 gene confers both drought tolerance and NaHCO₃ tolerance on tobacco. To determine whether the AfRAP2 gene also confers NaHCO₃ tolerance to woody plants, we obtained transgenic Populus davidiana×P. alba var. pyramidalis plants expressing the AfRAP2 gene. We subjected perforated leaves from the transgenic Populus davidiana×P. alba var. pyramidalis plants to NaHCO₃ stress. Similar to the overexpression of SlWRKY28 [40], WRKY33 [41], and PuPP2C [42], this gene can also enhance plant tolerance to salinity and alkalinity by participating in the oxidative stress response. The results showed that the damage to the leaves of wild-type Populus davidiana×P. alba var. pyramidalis was significantly greater than that to the leaves of the transgenic Populus davidiana×P. alba var. pyramidalis, indicating that the AfRAP2 gene confers alkaline salt tolerance to Populus davidiana×P. alba var. pyramidalis.

Conclusion

The results of this study indicate that under drought and saline-alkali stress treatments, the growth of transgenic plants was superior to that of wild-type plants. Physiological measurements further confirmed that the AfRAP2 gene plays a positive role in responding to drought and saline-alkali stress. This study demonstrates that AfRAP2 functions as a positive regulator in drought and saline-alkali stress responses, thereby contributes to the functional characterization of AP2/ERF transcription factors and laying the foundation for future mechanistic studies of AfRAP2.

Abbreviation

CAT

Catalase

SOD

Superoxide dismutase

POD

Peroxidase

AP2/ERF

APETALA2/Ethylene responsive factor

ERF

Ethylene responsive factor

DREB

Dehydration-responsive element binding protein

MDA

Malondialdehyde

Authors’ contributions

YY designed the study and wrote the manuscript. GQJ provided guidance for the experiments. LZX reviewed and revised the manuscript. Everyone participated in the peer review of the article. All authors read and approved the final manuscript.

Funding

Project on the Cloning of Salt- and Alkali-Tolerance Genes in Cold-Region Japonica Rice and the Development of Genetic Resources (2025ZX04B02-3), a major project funded by the Heilongjiang Provincial Department of Science and Technology with a budget of 200,000 yuan, December 30, 2025 – December 29, 2028.

Data availability

The datasets generated and analysed during the current study are available in the NCBI repositoryLOCUS ID: MT13552. The use of data and materials has been authorized by this experiment.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.