MfERF053 synergistically enhances drought resistance in Medicago sativa L. by regulating ABA signaling, antioxidant defense, and photosynthetic protection: a molecular mechanism study

MfERF053 synergistically enhances drought resistance in Medicago sativa L. by regulating ABA signaling, antioxidant defense, and photosynthetic protection: a molecular mechanism study | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 28 October 2025 V1 Latest version Share on MfERF053 synergistically enhances drought resistance in Medicago sativa L. by regulating ABA signaling, antioxidant defense, and photosynthetic protection: a molecular mechanism study Authors : Wei Duan , Qian Li [email protected] , Chun Liu , Xueli Zhang , Lijun Liu , Yongli Ran , Yuxiang Wang , Yaling Liu , and Wanjun Zhang 0000-0001-5806-3352 Authors Info & Affiliations https://doi.org/10.22541/au.176164413.38147054/v1 224 views 112 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Drought stress severely affects the growth and yield of alfalfa, and deciphering its drought-resistant molecular mechanisms is crucial for breeding drought-tolerant varieties. This study focused on the MfERF053 gene from Medicago falcata . By constructing overexpression (OE) and RNA interference (RNAi) lines, combined with physiological phenotype analysis, antioxidant enzyme activity determination, photosynthetic parameter detection, and transcriptome sequencing, the function and regulatory network of MfERF053 in drought response were revealed. The results showed that MfERF053 enhanced drought resistance of Medicago sativa through multiple mechanisms: In terms of stomatal regulation, it reduced leaf water loss rate by promoting stomatal closure to maintain water balance; in the antioxidant defense process, it increased the activities of catalase (CAT) and peroxidase (POD), thereby reducing the accumulation of reactive oxygen species (ROS) and alleviating membrane lipid peroxidation damage; in photosynthesis protection, it slowed down chlorophyll degradation, maintained the efficiency of photosystem II, and improved water use efficiency. Transcriptome analysis results indicated that MfERF053 was significantly enriched in the pathways of ”plant hormone signal transduction”, ”oxidoreductase activity”, and ”photosynthetic process”. It synergistically enhanced drought resistance by regulating the expression of genes involved in the ABA signaling pathway (such as the PYR / PYL - SnRK2 cascade), antioxidant genes (such as CAT1 and APX7 ), and photosynthesis-related genes (such as FTSH6 and PPH / PAO ). This study confirmed that MfERF053 is a positive regulator of alfalfa’s drought response, providing key genetic resources and theoretical basis for molecular breeding of drought-resistant alfalfa. MfERF053 synergistically enhances drought resistance in Medicago sativa L. by regulating ABA signaling, antioxidant defense, and photosynthetic protection: a molecular mechanism study Wei Duan 1 , Qian Li* 1 , Chun Liu 1 , Xueli Zhang 1 , Lijun Liu 1 , Yongli Ran 1 , Yuxiang Wang 1 , Yaling Liu 3 Wanjun Zhang 123 1 Key Laboratory of Grassland Resources and Ecology in Arid and Desert Areas of Western China, Ministry of Education / College of Grassland Science, Xinjiang Agricultural University, Urumqi 830052, Xinjiang, China 2 College of Grassland Science and Technology, China Agricultural University, Beijing 100193, China 3 National Center of Pratacultural Technology Innovation (Under Preparation), Hohhot 010010, China Abstract Drought stress severely affects the growth and yield of alfalfa, and deciphering its drought-resistant molecular mechanisms is crucial for breeding drought-tolerant varieties. This study focused on the MfERF053 gene from Medicago falcata . By constructing overexpression (OE) and RNA interference (RNAi) lines, combined with physiological phenotype analysis, antioxidant enzyme activity determination, photosynthetic parameter detection, and transcriptome sequencing, the function and regulatory network of MfERF053 in drought response were revealed. The results showed that MfERF053 enhanced drought resistance of Medicago sativa through multiple mechanisms: In terms of stomatal regulation, it reduced leaf water loss rate by promoting stomatal closure to maintain water balance; in the antioxidant defense process, it increased the activities of catalase (CAT) and peroxidase (POD), thereby reducing the accumulation of reactive oxygen species (ROS) and alleviating membrane lipid peroxidation damage; in photosynthesis protection, it slowed down chlorophyll degradation, maintained the efficiency of photosystem II, and improved water use efficiency. Transcriptome analysis results indicated that MfERF053 was significantly enriched in the pathways of ”plant hormone signal transduction”, ”oxidoreductase activity”, and ”photosynthetic process”. It synergistically enhanced drought resistance by regulating the expression of genes involved in the ABA signaling pathway (such as the PYR / PYL - SnRK2 cascade), antioxidant genes (such as CAT1 and APX7 ), and photosynthesis-related genes (such as FTSH6 and PPH / PAO ). This study confirmed that MfERF053 is a positive regulator of alfalfa’s drought response, providing key genetic resources and theoretical basis for molecular breeding of drought-resistant alfalfa. KEYWORDS Drought stress; MfERF053 gene; alfalfa; drought resistance; molecular mechanism 1 INTRODUCTION Drought stress is one of the major abiotic stresses restricting global agricultural production, severely affecting crop yield and quality (D’Odorico et al., 2021). The perennial leguminous forage alfalfa ( Medicago sativa L.), with its high protein content and rich nutritional value (containing essential amino acids, vitamins, and minerals), has become a core feed source in animal husbandry (Wolabu, 2020). However, under drought stress, alfalfa faces bottlenecks such as stunted growth, reduced biomass, and quality degradation (Bao et al., 2016; Luo et al., 2019). Deciphering its drought-resistant molecular mechanisms and exploring key regulatory genes are of great theoretical and application value for breeding drought-tolerant varieties (Ma et al., 2025; Luo et al., 2022). Plants have evolved complex drought-resistant regulatory networks during long-term evolution (Zhang et al., 2018; Cosme, 2023), and transcription factors play a core role by coordinating the expression of downstream genes (Nakano et al., 2006). The AP2/ERF family, a plant-specific superfamily of transcription factors, widely participates in hormone signal transduction, growth and development, and abiotic stress response by recognizing cis-elements such as GCC-box and DRE/CRT (Ohme-Takagi et al., 1995; Xu et al., 2008; Gasch et al., 2016). Recent studies have shown that ERF genes synergistically enhance drought tolerance through multi-dimensional mechanisms: At the level of hormone signaling, they cross-regulate abscisic acid (ABA), brassinosteroid (BR), and ethylene pathways. For example, TINY in Arabidopsis thaliana inhibits the phosphorylation of BIN2 kinase in the BR signaling pathway, relieves the inhibition of drought-responsive genes, and activates NCED3 to promote ABA synthesis, thereby regulating stomatal closure (Xie et al., 2019); PalERF2 in poplar ( Populus spp.) induces the expression of PalRD20 and PalSAG113 through the ethylene pathway (Chen et al., 2022); OsERF48 in rice ( Oryza sativa ) regulates OsCML16 through the calcium signaling pathway to enhance root development (Harin et al., 2017). In oxidative stress defense, ERF transcription factors scavenge reactive oxygen species (ROS) by regulating the activity of antioxidant enzymes. For example, in maize ( Zea mays ), overexpression lines of ZmEREBP60 show increased activity of peroxidase (POD), along with decreased contents of hydrogen peroxide (H₂O₂) and malondialdehyde (MDA) (Zhu et al., 2022); in poplar, ERF194 enhances the scavenging capacity of superoxide anions by upregulating redox enzyme genes (Huan et al., 2023). In terms of osmotic regulation and secondary metabolism, ERF regulates the synthesis of proline, soluble sugars, and anthocyanins. For example, overexpression of GmERF3 in soybean ( Glycine max ) increases the levels of proline and soluble carbohydrates in transgenic tobacco (Nicotiana tabacum) (Zhang et al., 2009); MdERF38 in apple ( Malus domestica ) interacts with MdMYB1 to promote anthocyanin synthesis in response to drought (An et al., 2020). In photosynthesis protection, ERF improves photosynthetic efficiency by maintaining the stability of photosystems and carbon metabolism. For example, overexpression of OsERF83 in rice increases photochemical efficiency under drought (Jung et al., 2021); heterologous expression of TSRF1 in tomato ( Solanum lycopersicum ) up-regulates photosynthetic genes in rice (Quan et al., 2010). As a core mechanism of drought tolerance, stomatal regulation is mediated by ERF through ABA-dependent/independent pathways. For example, TINY in Arabidopsis promotes stomatal closure through the ABA pathway (Xie et al., 2019), and overexpression of SlERF84 in tomato enhances ABA sensitivity to promote stomatal closure (Li et al., 2018). In addition, AtRAP2.4 in Arabidopsis activates epidermal wax synthesis genes to reduce transpiration rate and delay chlorophyll degradation (Yang et al., 2020), revealing the synergistic regulation of ERF on stomatal behavior and photosynthetic protection. Although research on the mechanisms of the ERF family in model plants and some crops has made progress, the regulatory network and multi-pathway synergistic mechanisms of ERF genes in non-model crops such as alfalfa have not been systematically analyzed. Previous studies found that the MfERF053 gene from Medicago falcata is significantly up-regulated under drought stress, and its overexpression in Arabidopsis shows enhanced root development, up-regulated expression of stress-resistant genes, and improved drought resistance (Li et al., 2022), but its molecular mechanism remains unclear. In this study, by constructing MfERF053 overexpression and RNAi lines of alfalfa, the physiological responses and transcriptome changes under drought stress were systematically analyzed, aiming to clarify its drought-resistant regulatory network and provide a theoretical basis for molecular breeding of alfalfa. 2 MATERIALS AND METHODS 2.1 Plant materials and growth environment ”Zhongmu No. 1” Medicago sativa was used as the experimental material. Plants were cultivated in a greenhouse under the following conditions: 16/8 h light/dark cycle, temperature of 25°C, and average light intensity of 400 μmol m⁻²·s⁻¹. Asexual propagation was performed via cuttings. Alfalfa plants with a height of approximately 40 cm were cut into 5 cm branches. Leaves were removed, while incomplete leaves and axillary buds were retained. The branches were inserted into a soil substrate infiltrated with Hoagland nutrient solution, and the substrate was compacted to ensure the base of the plants was fully exposed to nutrients, followed by incubation for rooting and regeneration. Nutrient solution was continuously supplemented during plant growth. Subsequently, the cuttings were transplanted into 80×90 mm pots. When the plants grew to a relatively uniform size (approximately 20 cm in height), at least 6 pots of plants per line were selected to initiate the natural drought stress experiment. After ensuring the soil water content was saturated, watering was ceased until the plants exhibited obvious drought stress symptoms. The soil substrate consisted of a well-mixed mixture of nutrient soil, vermiculite, and water-regulating stone at a ratio of 3:1:1 (v:v:v). 2.2 Vector construction and alfalfa transformation The full-length coding sequence (CDS) of MfERF053 had been previously cloned from wild Medicago falcata . To construct the overexpression and RNA interference (RNAi) vectors for this gene, the CDS of MfERF053 was inserted into the pCAMBIA1320-Flag and PEG100-RNAi vectors (driven by the CaMV35S promoter), respectively. Primers for transgenic alfalfa were designed: forward primer MfERF053 -F (5’-ACCTTCATCACCACCTCCAACAG-3’) and reverse primer MfERF053 -R (5’-CTCCTTCTCCGCCTTCACTCAC-3’). All recombinant vectors were introduced into Agrobacterium tumefaciens strain EHA105 via the freeze-thaw transformation method, and then used to transform leaf explants of ”Zhongmu No. 1” alfalfa to obtain transgenic plants. Information for all primers used in this study is listed in Table S1. 2.3 RNA extraction and RT-qPCR analysis Total RNA from each sample was extracted using the TRIzol kit (Sangon Biotech, Shanghai, China). First-strand cDNA was synthesized using the FastQuant RT kit containing gDNase (Tiangen Biotech, Beijing, China). RT-qPCR reactions were performed on an ABI 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) using 2×SG Fast qPCR Master Mix (Sangon Biotech). Transgenic alfalfa plants were identified via the identification of positive lines and RT-qPCR analysis (see Supplementary Figure S1; primers are listed in Supplementary Table S1). Three overexpression (OE) lines (named OE13, OE23, and OE70) and three RNAi lines (named RNAi07, RNAi13, and RNAi14) were selected for subsequent stress resistance analysis. 2.4 RNA-Seq and transcriptome data analysis 25-day-old well-grown plants of each line were subjected to natural drought stress, with plants grown under normal conditions serving as non-stressed controls. Leaves and roots of each line were collected and quickly frozen in liquid nitrogen. A total of 36 samples were included, covering WT, OE, and RNAi lines under both control and drought stress treatments, with 3 biological replicates per sample. Samples were sent to Denovo Gene Biotechnology Co., Ltd. (Guangzhou, China) for high-throughput sequencing. Raw reads were processed using fastp to remove low-quality sequences and generate clean reads, which were then mapped to the reference genome. Differentially expressed genes (DEGs) were identified with |log₂FC| ≥ 1 and FDR < 0.05 by calculating FPKM values. Functional enrichment of DEGs in GO and KEGG pathways was further analyzed. The top 10 GO terms and pathways ranked by FDR value were visualized in enrichment bubble plots, and DEGs enriched in relevant pathways were displayed in heatmaps (https://www.omicshare.com/). The sequencing data have been deposited in the Genome Sequence Archive (GSA) of the National Center for Bioinformation (CNCB) with the accession number CRA031490, and readers can access and retrieve the data via this accession number on the GSA platform (https://ngdc.cncb.ac.cn/gsa/). To validate the RNA-seq results, DEGs annotated in relevant pathways were selected for RT-qPCR verification. Primers were designed using the Sangon Biotech website (https://store.sangon.com/newPrimerDesign). The relative expression levels of 12 DEGs were detected by qRT-PCR, and the primer sequences are listed in Table S1. 2.5 Measurement of leaf water loss rate Trifoliate leaves with similar shape and size between the second and third internodes of each line were collected. Leaves were weighed immediately, placed on filter paper on a clean laboratory bench (40% RH, 24°C), and weighed hourly for a total of 8 hours. Each line was replicated 5 times. Relative water loss was calculated based on the initial weight of the leaves, with the initial water loss rate of each leaf set to 0 (Luo et al. 2022). 2.6 Measurement of relative water content (RWC) Under normal and drought stress conditions, leaves with similar shape and size between the second and third internodes of each line were collected. After measuring the fresh weight (Wf), the leaves were immersed in distilled water in a sealed tube at room temperature for 4 hours, then incubated at 5°C overnight. The turgid weight (Wt) was measured the next day. Subsequently, the samples were wrapped in tin foil and dried at 80°C to a constant weight (approximately 72 hours), defined as the dry weight (Wd). RWC was calculated as follows: RWC = (W f - W d )/(W t - W d ) (Zhang et al. 2024). Each line was replicated 5 times. 2.7 Measurement of relative conductivity Under normal and drought stress conditions, leaves with similar shape and size between the second and third internodes of each line were collected. A 0.1 g sample (free of impurities) was placed in a 25 mL test tube, and 20 mL of deionized water was added to submerge the sample. The tube was allowed to stand for 3 hours, with shaking every 30 minutes during incubation. Conductivity (C1) was measured with a conductivity meter at a constant temperature of 20~25°C. The sample was then heated in a boiling water bath at 100°C for 10 minutes, cooled to room temperature, and the boiled conductivity (C2) was measured at 20~25°C. Relative leaf conductivity (%) was calculated as: (C 1 /C 2 ) × 100% (Tao et al. 2022). Each line was replicated 5 times. 2.8 Measurement of stomatal density and aperture Under normal and drought stress conditions, the third fully expanded leaf at the top of each line was collected to observe stomatal density and aperture (Lv et al. 2023). A drop of water was placed on a glass slide, and the epidermis near the main vein on the abaxial surface of the leaflet was gently peeled off with tweezers, spread on the water drop, flattened with a coverslip, and excess water was absorbed with filter paper. Stomata were photographed using a stereomicroscope (Nikon, Tokyo, Japan) and counted using ImageJ software (http://imagej.nih.gov/ij/). Stomatal aperture (width:length ratio) was measured via software: an aperture ratio ≤ 0.2 was defined as fully closed; 0.3-0.5 as partially open; and ≥ 0.5 as fully open (Wu et al. 2023). Each line was replicated 10 times, with at least 10 clear stomata per microscopic field. 2.9 Measurement of physiological indicators The activities of catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD), as well as the contents of malondialdehyde (MDA), hydrogen peroxide (H₂O₂), and superoxide anion (O₂⁻), along with DAB and NBT staining, were detected using corresponding kits (Solarbio Science & Technology Co., Ltd., Beijing, China). Each line was replicated 3 times. 2.10 Measurement of chlorophyll pigment content Fresh leaves were harvested, and 0.05 g of each sample was weighed. The weighed fresh leaves were cut into pieces, placed in a test tube, and immersed in 10 mL of extraction solution (ethanol:acetone:distilled water = 4.5:4.5:1, v/v/v) for pigment extraction. The extract was centrifuged, and the absorbance of the supernatant at 663 nm, 646 nm, and 470 nm was measured using a spectrophotometer (Chen et al. 2010). Each line was replicated 4 times. The concentrations of chlorophyll a/b and carotenoids were calculated as follows: Chlorophyll a concentration (mg·L⁻¹) = 12.21×OD₆₆₃ - 2.81×OD₆₄₆ Chlorophyll b concentration (mg·L⁻¹) = 20.13×OD₆₄₆ - 5.03×OD₆₆₃ Carotenoid concentration (mg·L⁻¹) = (1000×OD₄₇₀ - 3.27×Ca - 104×Cb)/229 2.11 Determination of photosynthetic parameters A Ciras-4 portable photosynthesis system was used to measure the net photosynthetic rate (Pn), transpiration rate (E), stomatal conductance (Gs), and intercellular CO₂ concentration (Ci) of alfalfa. Water use efficiency (WUE) was calculated as: WUE = Pn/E. Each line was replicated 7 times. 2.12 Measurement of chlorophyll fluorescence Leaves with similar shape and size between the second and third internodes of each line were selected, and chlorophyll fluorescence was measured using a PlantExplorer XS plant photosynthetic phenotype measurement system (Huinuo Ruide (Beijing) Technology Co., Ltd., Beijing, China). Prior to each measurement, leaves were dark-adapted for 30 minutes. The maximum quantum yield of photosystem II ( F v / F m ) was calculated as ( F m - F o )/ F m , where Fo is the minimum fluorescence yield obtained with a weak measuring beam, and Fm is the maximum fluorescence yield estimated after applying a saturation pulse to quench the reaction centers. Additionally, the variable chlorophyll fluorescence ( F v ) was estimated as F m - F o . Each leaf was then exposed to photosynthetically active radiation for 5 minutes. Under light-adapted conditions, the steady-state fluorescence (Fs) and maximum fluorescence (Fm’) were quantified using the aforementioned saturation pulse. Non-photochemical quenching ( NPQ ) was calculated as: NPQ = F s / F m ’ - F s / F m . Each line was replicated at least 25 times. 2.13 Statistical analysis Statistical analysis was performed using SPSS 27.0 software, and graphs were generated using GraphPad Prism 9.5.1 software. Data were obtained from at least 3 independent replicates, with error bars representing standard deviation. Different letters above the bar charts indicate significant differences between different lines under the same treatment, as determined by one-way analysis of variance (ANOVA) and the least significant difference (LSD) test ( P < 0.05). Different * above the bar charts indicate significant differences in the same line between different treatments, as determined by one-way ANOVA and LSD test ( P < 0.05, P < 0.01, P < 0.001). 3 RESULTS 3.1 MfERF053 is a positive regulator of alfalfa drought response The regulatory role of MfERF053 in drought stress was analyzed using overexpression (OE) and RNA interference (RNAi) alfalfa lines. A total of 72 OE lines and 21 RNAi lines were generated. PCR and RT-qPCR screening showed that 40 OE lines and 10 RNAi lines were positive, expressing MfERF053 at different mRNA levels (Figure S1). After 5 days of natural drought stress, leaves of RNAi lines showed severe curling and wilting, wild-type (WT) plants exhibited mild leaf curling and wilting, while OE lines grew well. With the further aggravation of drought stress, RNAi lines died first, followed by WT plants, and OE lines died last (Figure 1a, b). On the 13th day of drought stress, the survival rate of RNAi lines (12.96%) was significantly lower than that of OE lines (68.05%) (Figure 1c). In addition, under drought stress, the relative water content (RWC) of alfalfa leaves significantly decreased; the RWC of OE lines (29.45%) was significantly higher than that of WT plants (16.15%), while the RWC of WT plants was significantly higher than that of RNAi lines (7.87%) (Figure 1d). Both relative conductivity (RC) and malondialdehyde (MDA) content increased; the RC and MDA content of RNAi lines (22.51%, 180.03 nmol·g -1 ) were significantly higher than those of WT plants (13.03%, 133.64 nmol·g -1 ), while those of WT plants were significantly higher than those of two OE lines (OE13, OE70) (7.33%, 103.20 nmol·g -1 ) (Figure 1e, f). Under drought stress, the damage degree of OE lines was significantly lower than that of RNAi lines, confirming that MfERF053 positively regulates alfalfa drought resistance by enhancing water retention capacity and alleviating membrane lipid peroxidation damage. Regarding the root system, after normal and drought stress treatments (10 days of stress), the overexpression (OE) lines had more lateral roots and greater root weight than the RNA interference (RNAi) lines (Supplementary Figure 2a,c). Secondly, under drought stress treatment, the relative water content (RWC) and root weight of alfalfa roots both decreased significantly; the RWC and root weight of the root system in OE lines (11.62%, 0.51 g) were significantly higher than those in RNAi lines (3.72%, 0.21 g) (Figure S2b,c). These results indicate that MfERF053 promotes lateral root development and enhances water absorption capacity. In conclusion, MfERF053 is a positive regulator of alfalfa’s drought response. FIGURE 1 MfERF053 is a positive regulator of alfalfa drought response. (a, b) Phenotypes of RNAi, WT, and OE alfalfa lines under natural drought stress. (c) Mortality rate of each line after 13 days of natural drought. (d-f) Relative water content (d), relative conductivity (e), and malondialdehyde (MDA) content (f) in leaves of each line under well-watered and drought stress treatments. Error bars represent the mean ± standard deviation (SD) of at least three independent replicates. Different lowercase and uppercase letters indicate statistically significant differences under well-watered and drought stress treatments, respectively ( p < 0.05). *, **, and *** indicate statistically significant differences compared with the control group at p < 0.05, p < 0.01, and p < 0.001, respectively; ns indicates no significant difference. 3.2 MfERF053 positively regulates stomatal movement to modulate water loss from alfalfa leaves An air-drying experiment was performed for 8 hours at room temperature using detached leaves from plants of each line. Leaves of RNAi lines exhibited slight wilting symptoms after 2 hours, while the phenotypes of leaves from WT and OE lines remained normal; with the passage of time, the wilting symptoms of RNAi line leaves were the most severe (Figure 2a). During the entire 8-hour dynamic dehydration process, OE line leaves retained the most water (54.15%), followed by WT plant leaves (38.15%), and RNAi line leaves retained the least water (9.22%) (Figure 2b). To explain how OE line leaves can retain more water, we analyzed the stomatal density, stomatal aperture, and stomatal regulation of the abaxial epidermis of leaves from RNAi, WT, and OE alfalfa lines (Figure 2c-g). There was no significant difference in stomatal density among leaves of different lines (Figure 2c). Under normal treatment, the average stomatal aperture (width: length) of OE lines (0.32) was significantly lower than that of RNAi lines (0.39) (Figure 2d,e). Under drought stress treatment, the average stomatal aperture of all lines decreased significantly; the average stomatal aperture of OE lines (0.20) was significantly lower than that of WT plants (0.25), and the average stomatal aperture of WT plants was significantly lower than that of RNAi lines (0.30) (Figure 2d,e). In addition, under normal treatment, there was no obvious difference in stomatal opening and closing status (Figure 2f); however, under drought stress treatment, the number of fully closed stomata in OE lines (35.80%) was higher than that in WT plants (23.59%) and RNAi lines (9.98%), and the number of fully open stomata in RNAi lines (44.89%) was greater than that in WT plants (28.26%) and OE lines (19.76%) (Figure 2g). These results indicate that MfERF053 may regulate stomatal closure, thereby modulating leaf water loss under drought stress. FIGURE 2 MfERF053 positively regulates stomatal closure in alfalfa under drought stress. (a) Phenotypes of detached leaves from each line after 8 hours of air-drying. (b) Water loss rate of detached leaves. (c) Stomatal density of each line. (d) Stomatal phenotypes of RNAi, WT, and OE alfalfa lines under natural drought stress. (e) Stomatal conductance of each line under normal and drought stress treatments. (f, g) Percentages of three types of stomata in alfalfa leaves under normal (f) and drought stress (g) treatments. Error bars represent the mean ± standard deviation (SD) of at least three independent replicates. Different lowercase and uppercase letters indicate statistically significant differences under normal and drought stress treatments, respectively ( p < 0.05). *, **, and *** indicate statistically significant differences compared with the control group at p < 0.05, p < 0.01, and p < 0.001 levels, respectively; ns indicates no significant difference. 3.3 MfERF053 positively regulates enzyme activities to scavenge ROS under drought stress To evaluate the role of MfERF053 in drought-induced oxidative stress, the degree of leaf damage in each line was determined using NBT and DAB staining, and the contents of superoxide anion (O₂⁻) and hydrogen peroxide (H₂O₂) were measured (Figure 3). Results showed that under drought stress treatment, the contents of O₂⁻ and H₂O₂ in leaves of all lines increased significantly (Figure 3a-d). The NBT and DAB staining levels in leaves of RNAi lines were higher than those of WT and OE lines (Figure 3a,b); the contents of O₂⁻ and H₂O₂ in RNAi lines (1043.50 nmol·g⁻¹, 6.94 μmol·g⁻¹) were significantly higher than those in WT plants (847.47 nmol·g⁻¹, 5.72 μmol·g⁻¹), and those in WT plants were significantly higher than those in OE lines (628.82 nmol·g⁻¹, 4.61 μmol·g⁻¹) (Figure 3c,d). These results indicate that MfERF053 can reduce cellular damage and enhance plant resistance to drought stress by decreasing the accumulation of reactive oxygen species (ROS) under drought stress. FIGURE 3 MfERF053 positively regulates antioxidant defense in alfalfa under drought stress. (a, b) NBT (a) and DAB (b) staining of RNAi, WT, and OE lines under normal and drought stress treatments. (c, d) Contents of superoxide anion (O₂⁻) (c) and hydrogen peroxide (H₂O₂) (d). (e-g) Activities of catalase (CAT) (e), peroxidase (POD) (f), and superoxide dismutase (SOD) (g) in each line under normal and drought stress treatments. Error bars represent the mean ± standard deviation (SD) of three independent replicates. Different lowercase and uppercase letters indicate statistically significant differences under normal and drought stress treatments, respectively ( p < 0.05). *, **, and *** indicate statistically significant differences compared with the control group at p < 0.05, p < 0.01, and p < 0.001 levels, respectively; ns indicates no significant difference. To further reveal the mechanism by which MfERF053 regulates ROS accumulation, the activities of key enzymes in the antioxidant defense system were analyzed (Figure 3). Under drought stress treatment, the activities of catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) in all lines increased significantly (Figure 3e-g), among which the increase in enzyme activity of OE lines (105.61%, 324.69%, 137.53%) was significantly higher than that of RNAi lines (44.23%, 130.70%, 88.07%). These results suggest that MfERF053 may enhance ROS scavenging capacity by upregulating antioxidant enzyme activities, thereby effectively alleviating drought stress-induced oxidative damage and improving plant drought tolerance. 3.4 MfERF053 positively regulates photosynthetic characteristics of alfalfa under drought stress Drought stress typically inhibits photosynthesis. To confirm whether MfERF053 affects the photosynthetic efficiency of alfalfa under drought stress, analyses were performed from three aspects: chlorophyll fluorescence, chlorophyll content, and photosynthetic parameters (Figure 4). Regarding chlorophyll fluorescence, under drought stress treatment, the F v / F m (maximum photochemical efficiency of photosystem II) of leaves in all lines decreased significantly (Figure 4a,c); the decrease magnitude of OE lines (3.18%) was significantly lower than that of WT plants (21.66%), and the decrease magnitude of WT plants was significantly lower than that of RNAi lines (34.68%). The NPQ (non-photochemical quenching) of leaves in all lines increased (Figure 4b,d), with RNAi lines showing the largest increase (265.80%), which was significantly higher than that of WT plants (197.53%) and OE lines (23.63%). These results indicate that MfERF053 may improve drought-resistant photosynthetic performance by maintaining photosystem II efficiency and light energy balance. In terms of chlorophyll content, under drought stress treatment, the contents of chlorophyll a and chlorophyll b in all lines decreased significantly (Figure 4e,f), and the decrease extent of RNAi lines (54.39%, 56.56%) was significantly higher than that of OE lines (11.28%, 19.47%). Under normal treatment, the carotenoid content of RNAi lines (1.81 mg·g⁻¹) was significantly higher than that of WT plants (0.37 mg·g⁻¹) and OE lines (0.39 mg·g⁻¹) (Figure 4g); under drought stress treatment, the carotenoid content of RNAi lines (1.37 mg·g⁻¹) decreased significantly, while that of WT plants (0.55 mg·g⁻¹) and OE lines (0.66 mg·g⁻¹) increased significantly. However, the carotenoid content of RNAi lines remained significantly higher than that of WT plants and OE lines. These results suggest that MfERF053 may enhance alfalfa’s drought resistance by maintaining chlorophyll stability and regulating the dynamic response of carotenoids. For photosynthetic parameters, under drought stress treatment, the Pn (net photosynthetic rate), Tr (transpiration rate), Gs (stomatal conductance), and Ci (intercellular CO₂ concentration) of all lines decreased significantly (Figure 4h-k). Under drought stress, the decrease magnitude of Pn in RNAi lines (68.83%) was significantly higher than that in OE lines (9.69%) (Figure 4h); whereas the decrease magnitudes of Tr, Gs, and Ci in OE lines (44.93%, 50.85%, 40.35%) were significantly higher than those in RNAi lines (34.06%, 35.71%, 34.46%) (Figure 4i-k). In addition, under normal treatment, the WUE (water use efficiency) of RNAi lines (3.66 mmol CO₂ ·m⁻²·s⁻¹ per mol H₂O ·m⁻²·s⁻¹) was significantly lower than that of OE lines (6.41 mmol CO₂ ·m⁻²·s⁻¹ per mol H₂O ·m⁻²·s⁻¹) (Figure 4l); under drought stress treatment, the WUE of RNAi lines and WT plants decreased significantly, while that of OE lines increased significantly (Figure 4l). These results indicate that MfERF053 may improve photosynthetic adaptability under drought by coordinating stomatal and non-stomatal limiting factors. In summary, MfERF053 helps maintain the photosynthetic capacity of alfalfa under drought stress. FIGURE 4 Photosynthetic characteristics of MfERF053 -OE and RNAi alfalfa lines under drought stress. (a, b) Phenotypes of F v / F m (a) and NPQ (b) in RNAi, WT, and OE lines under normal and drought stress treatments. (c, d) F v / F m (c) and NPQ (d) values. (e-g) Contents of chlorophyll a (e), chlorophyll b (f), and carotenoid (g) in each line under normal and drought stress treatments. (h-l) Net photosynthetic rate (Pn) (h), transpiration rate (Tr) (i), stomatal conductance (Gs) (j), intercellular CO₂ concentration (Ci) (k), and water use efficiency (WUE) (l) in each line under normal and drought stress treatments. Error bars represent the mean ± standard deviation (SD) of at least three independent replicates. Different lowercase and uppercase letters indicate statistically significant differences under normal and drought stress treatments, respectively ( p < 0.05). *, **, and *** indicate statistically significant differences compared with the control group at p < 0.05, p < 0.01, and p < 0.001 levels, respectively; ns indicates no significant difference. 3.5 Transcriptome analysis of RNAi, WT, and OE alfalfa lines under drought stress 3.5.1 Transcriptome sequencing To further investigate the molecular mechanism by which MfERF053 confers drought resistance, RNA sequencing (RNA-seq) was performed on leaf and root samples from RNAi, WT, and OE lines under normal and drought stress treatments for 5 days, yielding a total of 1,756,552,170 clean reads. Data quality assessment showed that Q30 values of all samples exceeded 95%, with the minimum value being 95.49% and the maximum 97.65%. The GC content was similar across all samples, ranging from 41.63% to 42.38%. Sequence alignment of clean reads from each sample with the reference genome resulted in alignment efficiencies ranging from 80.35% to 93.56% (Table S2). These results indicate the accuracy and reliability of the sequencing process, and the sequencing data meet the requirements for subsequent analyses. In the results of transcriptome PCA analysis (Figure S3a-c), samples from different lines (RNAi, WT, OE) and condition groups (leaf L, leaf under drought stress LD, root R, root under drought stress RD) showed distinct intra-group clustering and inter-group separation. To explore the effect of drought stress on the transcriptome of each line, a two-way comparative analysis of gene expression profiles was conducted, with leaves or roots after 5 days of natural drought stress as the experimental group and leaves or roots under normal growth conditions as the control group. In leaf samples, 6771/6787 upregulated/downregulated DEGs (differentially expressed genes) were identified under RNAi-L-vs-RNAi-LD, 3383/5248 under WT-L-vs-WT-LD, and 5385/3430 under OE-L-vs-OE-LD. In root samples, 5523/5757 upregulated/downregulated DEGs were identified under RNAi-R-vs-RNAi-RD, 2301/2306 under WT-R-vs-WT-RD, and 3133/7440 under OE-R-vs-OE-RD (Figure S3d-f). 3.5.2 MfERF053 affects multiple pathways under drought stress A Venn diagram was constructed to screen for common DEGs regulated by MfERF053 across different lines and tissues under drought stress, laying the foundation for enrichment analysis and pathway exploration. In leaf sample analysis, there were 1652/1386 common upregulated/downregulated DEGs among RNAi lines, WT plants, and OE lines, while 3782/4268, 1832/511, and 659/1946 upregulated/downregulated DEGs in RNAi lines, WT plants, and OE lines, respectively, were specifically regulated by drought stress (Figure S4). In root sample analysis, 996/1279 common upregulated/downregulated DEGs were identified among RNAi lines, WT plants, and OE lines, whereas 3263/2479, 1178/3903, and 548/400 upregulated/downregulated DEGs in RNAi lines, WT plants, and OE lines, respectively, were specifically regulated by drought stress (Figure S5). In the combined analysis of leaves and roots, 333/74 common upregulated/downregulated DEGs were found among RNAi lines, WT plants, and OE lines (Figure 5). To further explore the mechanism by which MfERF053 affects drought stress tolerance, KEGG (Kyoto Encyclopedia of Genes and Genomes) and GO (Gene Ontology) enrichment analyses were performed on the common DEGs of each line after drought stress. The results showed that in leaf samples (Figure S4), 1652 upregulated DEGs were mainly enriched in KEGG pathways such as ”plant hormone signal transduction” and ”arginine and proline metabolism”, which may be involved in plant hormone regulation and osmotic adjustment in response to drought. Meanwhile, GO enrichment involved functions related to ”response to abiotic stimulus”, ”response to oxygen-containing compounds”, and ”thylakoid”, indicating that leaf tissues adapt to drought through these mechanisms. The 1386 downregulated DEGs were mainly associated with KEGG pathways such as ”photosynthesis-antenna proteins” and ”carbon fixation”, suggesting that drought inhibits the expression of photosynthesis-related genes in leaf tissues and affects photosynthetic processes; their GO-enriched functions such as ”ribosome” structural composition imply an impact on basic physiological processes like protein synthesis. FIGURE 5 KEGG and GO enrichment analyses of common differentially expressed genes (DEGs) in leaves and roots of RNAi lines, WT plants, and OE lines. The Y-axis represents KEGG or GO pathways; the X-axis represents the enrichment factor. High FDR values are indicated in red, and low FDR values in blue. The enrichment factor refers to the ratio of the number of differentially expressed genes to the total number of annotated genes in the pathway. The larger the enrichment factor, the higher the degree of enrichment. In root samples (Figure S5), 996 upregulated DEGs were enriched in KEGG pathways including ”arginine and proline metabolism” and ”metabolic pathways”, which may be involved in root substance metabolism and osmotic adjustment. GO enrichment in ”response to abiotic stimulus” and ”response to oxygen-containing compounds” indicates that root tissues respond to drought through similar mechanisms. The 1279 downregulated DEGs were mainly involved in KEGG pathways such as ”nitrogen metabolism” and ”plant MAPK signaling pathway”, which may affect nitrogen utilization and signal transduction in roots; GO-enriched functions like ”response to external stimulus” suggest that the physiological functions of root tissues are affected by drought. In the combined analysis of leaf and root samples (Figure 5), 333 upregulated DEGs were enriched in KEGG pathways such as ”insulin resistance” (annotation may be broad in plant studies) and ”arginine and proline metabolism”, with GO enrichment in functions including ”response to abiotic stimulus” and ”response to oxygen-containing compounds”. The 74 downregulated DEGs were mainly associated with KEGG pathways such as ”plant hormone signal transduction” and ”plant MAPK signaling pathway”, and GO enrichment involved functional pathways like ”abscisic acid binding”. Additionally, in the GO molecular function enrichment of upregulated DEGs, the ”oxidoreductase activity” pathway was significantly enriched (Figure S6). Pathways such as ”plant hormone signal transduction”, ”plant MAPK signaling pathway”, ”arginine and proline metabolism”, ”response to oxygen-containing compounds”, ”oxidoreductase activity”, and photosynthesis-related pathways were significantly enriched, covering signal transduction, substance metabolism, photosynthesis, and oxidative stress processes. This indicates that plants adapt to drought stress by regulating hormone signals, substance metabolism, photosynthesis, and responses to oxidative stress. MfERF053 may affect substance metabolism, photosynthesis, signal transduction, and stress responses by regulating the expression of genes related to these pathways in leaves and roots, thereby altering plant drought tolerance. 3.5.3 Expression pattern analysis of differentially expressed genes regulated by MfERF053 in plant hormone signal transduction, photosynthetic system, and oxidative stress pathways under drought stress From KEGG and GO pathway enrichment analyses, it was found that ”plant hormone signal transduction” (ABA), ”oxidoreductase activity”, and photosynthesis-related pathways were the most significantly enriched pathways. To further explore how MfERF053 enhances alfalfa’s drought stress resistance through regulation of these major pathways, RNA-seq data were used to detect the expression patterns of genes involved in ABA signal transduction, oxidoreductase activity, and photosynthetic processes (Figure 6). In the ABA signal transduction pathway, MfERF053 enhances ABA signal transmission by upregulating the ABA receptor gene PYR / PYL , kinase SnRK2 , and transcription factor ABF , while downregulating the negative regulator PP2C . Among them, PYR / PYL recognizes ABA through binding activity, relieves the inhibition of SnRK2 by PP2C , and activates the phosphorylation catalytic activity of SnRK2 to promote the expression of downstream stomatal closure-related genes, thereby reducing water transpiration (Figure 6a). In the oxidoreductase activity pathway, this gene significantly induces the expression of genes such as catalase CAT1 and ascorbate peroxidase APX , enhances catalytic activity to scavenge reactive oxygen species (ROS) accumulated under drought stress, reduces oxidative damage, and maintains intracellular redox balance (Figure 6b). In the photosynthetic process pathway, MfERF053 maintains photosynthetic efficiency through multi-pathway coordinated regulation. On the one hand, it downregulates the expression of chlorophyll-degrading enzyme genes PPH / PAO , inhibits drought-induced chlorophyll decomposition, slows the decline in light absorption efficiency, and ensures the basis for light energy capture. On the other hand, it upregulates the ELIP gene to bind excess light energy and reduce the loss of light absorption efficiency; meanwhile, it significantly upregulates the protease FTSH6 to accelerate the degradation of damaged PSII proteins, enhance PSII repair capacity, stabilize the electron transport chain, and alleviate photosynthetic apparatus damage. In addition, this regulatory pattern avoids the reduction of light-harvesting complexes caused by abnormal expression of genes such as LICB3 , and the abnormal assembly of photosynthetic systems caused by changes in genes such as PSBP1 / 4 , thereby maintaining the stability of photosynthetic apparatus and light energy transfer efficiency, ensuring carbon assimilation capacity under drought conditions, and ultimately improving alfalfa’s adaptability to drought stress (Figure 6c). MfERF053 enhances alfalfa’s adaptability to drought stress from the aspects of water regulation, cellular protection, and energy metabolism by coordinately regulating the expression of genes related to ABA signal transduction, redox homeostasis, and photosynthetic processes. FIGURE 6 Differentially expressed genes (DEGs) involved in ABA signal transduction, oxidoreductase activity, and photosynthetic process. (a) ABA signal transduction, (b) oxidoreductase activity, (c) photosynthetic process. To identify gene modules regulated by MfERF053 and responsive to drought, weighted gene co-expression network analysis (WGCNA) was performed on the common differentially expressed genes (DEGs) of each line in leaves and roots (Figure S7). A total of 3078 DEGs in leaves were divided into 7 modules (Figure S7a,b). After drought stress, the expression of genes in the purple module of OE lines was significantly higher than that in RNAi and WT lines (Figure S7c). Core genes in this module, such as ACX5 (containing ABRE and G-box elements, which can integrate hormones such as ABA and light signals to regulate fatty acid metabolism and enhance drought resistance), FTSH2 , PAO , and PPH (maintaining carbon assimilation under drought), played important roles (Figure S7g). In roots, 2294 DEGs were divided into 9 modules (Figure S7d,e). After drought stress, the expression of the green-yellow module in OE lines increased significantly (Figure S7f). Among them, core genes including SUS4 (regulating sugar metabolism to maintain osmotic pressure and enhance stress resistance, serving as a key node in sugar metabolism and stress resistance mechanisms) and GSTU (with conserved domains, acting as a hub connecting antioxidant defense, secondary metabolism, and stress signals) exerted a synergistic effect (Figure S7h), effectively improving plant adaptability under drought stress. To further verify the RNA-seq results, quantitative real-time PCR (qRT-PCR) analysis was performed, and the results also confirmed that these genes were regulated after drought stress treatment (Figure S8). 4 Discussion Under drought stress, maintaining water balance is crucial for plant survival. As key channels for gas and water exchange between plants and the external environment, stomata play a core role in regulating water balance through dynamic adjustments (Xiong et al. 2002). In this study, under drought stress, the proportion of fully closed stomata in MfERF053 -overexpressing (OE) plants was significantly increased, by 12.21% and 25.82% compared with wild-type (WT) and RNAi lines, respectively (Figure 2g), and the water loss rate of detached leaves was significantly reduced (Figure 2b). This phenotype is similar to the mechanism by which Arabidopsis TINY promotes stomatal closure via the ABA pathway (Xie et al. 2019), but MfERF053 is unique in that it achieves rapid and precise dynamic responses by enhancing stomatal sensitivity to ABA rather than altering stomatal density (Figure 2c). At the molecular mechanism level, transcriptome data showed that MfERF053 could upregulate the ABA receptor PYR / PYL and downstream kinase SnRK2.3 / 2.4 , while inhibiting the negative regulator PP2C (Figure 6a), thereby constructing an efficient ”signal perception-rapid closure” regulatory loop. This mechanism is highly consistent with the report that wheat TaPYL1-1B optimizes stomatal movement through ABA signaling (Mao et al. 2022), fully demonstrating that the ERF family has a conserved signal amplification mechanism in the stomatal regulatory pathway. Through this mechanism, plants can quickly perceive signals at the early stage of drought stress, close stomata in a timely manner to effectively reduce water loss, while maintaining a certain level of gas exchange to ensure basic photosynthesis (Daszkowska-Golec and Szarejko 2013; Agurla et al. 2018). This is because ABA, as an important signal molecule for plants to respond to adversity, rapidly accumulates under drought stress, binds to PYR / PYL receptors, relieves the inhibition of SnRK2 kinase by PP2C , and the activated SnRK2 kinase then phosphorylates downstream target proteins, prompting stomatal closure and reducing water loss (Li et al. 2024; Hsu et al. 2021). As an important organ for water absorption, root development directly affects plant drought tolerance (Smith and De 2012). Overexpression of MfERF053 significantly promoted lateral root development; under drought conditions, root weight increased by 2.43-fold and root relative water content increased by 2.12-fold compared with RNAi lines (Figure S2b,c). This phenomenon is consistent with the phenotype of Medicago falcata homologous genes promoting root elongation (Li et al. 2022), suggesting that MfERF053 may enhance water capture capacity in arid environments by expanding root surface area. This synergistic mechanism of ”aboveground stomatal closure reducing transpiration-underground root expansion increasing water absorption” allows OE lines to maintain a higher level of overall water balance under drought stress (Figure 1d). Unlike the strategy of maize ZmSDD1 improving water use efficiency by reducing stomatal density (Liu et al. 2015), MfERF053 focuses on the synergistic response of aboveground and underground organs, reflecting the diversified ”resource acquisition and conservation” adaptive strategies formed by plants in response to drought environments during evolution. This synergistic strategy helps plants acquire and preserve water more effectively under drought conditions, providing strong support for their survival and growth. Studies have shown that the increase and elongation of lateral roots can increase the contact area between roots and soil, improve water absorption efficiency (Ranjan et al. 2022; Wang et al. 2024), and root development may also affect the rhizosphere microbial community, further improving plant water acquisition capacity (Chen et al. 2022). Drought stress leads to massive accumulation of reactive oxygen species (ROS) in plants, triggering membrane lipid peroxidation and causing severe cellular damage (Mittler et al. 2004; Tang et al. 2013). Many plants can alleviate oxidative damage and osmotic stress caused by drought by activating antioxidant defense systems, enhancing antioxidant enzyme activities, and promoting the accumulation of compatible osmolytes such as soluble sugars and free proline (Gill and Tuteja 2010). Under drought conditions, MfERF053 -overexpressing plants exhibited significant ROS scavenging capacity. Their catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) activities were increased by 142.33%, 193.08%, and 134.56% compared with RNAi lines, respectively (Figure 4e-g), while H₂O₂ and MDA contents were decreased by 66.41% and 61.27%, respectively (Figure 3d, 1f). This result is highly consistent with the mechanism by which maize ZmEREBP60 reduces MDA content by enhancing POD activity (Zhu et al. 2022), strongly confirming that ERF family genes have a conserved function in maintaining cellular redox homeostasis (Mizoi et al. 2012). Transcriptome data further revealed that MfERF053 significantly upregulated antioxidant genes such as CAT1 and APX7 (Figure 6b). The enzymes encoded by these genes cooperate to efficiently scavenge superoxide anion (O₂⁻) and hydrogen peroxide (H₂O₂), thereby effectively reducing oxidative damage (Apel and Hirt 2004; Yoshimura and Ishikawa 2024). Notably, the regulation of glutathione S-transferase ( GSTU , Figure S7h) by MfERF053 may be involved in the detoxification of secondary metabolites. This functionally echoes the report that soybean GmNTF2B-1 reduces nuclear ROS by interacting with oxidoreductases (Chen et al. 2021), suggesting that the NTF2L family may have a conserved mechanism of action in antioxidant defense (Song et al. 2022). However, it remains unclear whether MfERF053 directly binds to the promoter regions of these genes (e.g., recognizing GCC-box elements) to regulate their expression, which needs to be verified by Electrophoretic Mobility Shift Assay (EMSA) to clarify the precise molecular mechanism of its transcriptional regulation. Previous studies have shown that ERF transcription factors can activate or inhibit gene expression by recognizing GCC-box elements in the promoters of target genes (Ohme-Takagi et al. 1995), but the binding of MfERF053 to the promoters of these antioxidant genes requires further experimental verification. Photosynthesis is the core process for plants to maintain energy supply under drought conditions (Talbi et al. 2020). MfERF053 maintains the stability of photosynthetic efficiency through a multi-pathway coordinated regulatory mechanism. On the one hand, this gene downregulates the expression of chlorophyll-degrading enzyme genes PPH / PAO (Figure 6b), reduces the corresponding enzyme activities, and effectively slows down the drought-induced chlorophyll decomposition process. Under drought stress, the decrease in chlorophyll a/b content was 43.11%/37.09% lower than that in RNAi lines, respectively (Figure 4e,f), showing cross-species similarity to the function of rice OsERF83 delaying chlorophyll decomposition by regulating photosynthetic antenna proteins (Jung et al. 2021). As a key pigment for light energy capture, chlorophyll stability is crucial for photosynthesis (Wang et al. 2021), and the regulation of PPH / PAO by MfERF053 directly maintains the relative stability of photosynthetic pigment content. On the other hand, MfERF053 upregulates the photosystem II repair protein gene FTSH6 (Figure 6c), resulting in a decrease of only 3.18% in the maximum quantum efficiency ( F v / F m ) of photosystem II (Figure 4c), significantly alleviating photosystem damage induced by strong light. Fluorescence parameters showed that the increase in non-photochemical quenching ( NPQ ) of OE lines was only 23.63%, much lower than 265.80% of RNAi lines (Figure 4d), indicating that they can allocate excess light energy more efficiently and avoid oxidative damage caused by excess light energy (Bassi and Dall’Osto 2021). From the perspective of transcriptome data, this precise regulatory pattern also avoids the reduction of light-harvesting complexes caused by abnormal expression of genes such as LICB3 and the abnormal assembly of photosynthetic systems caused by changes in genes such as PSBP1 / 4 , thereby maintaining the stability of photosynthetic apparatus and light energy transfer efficiency, and ensuring carbon assimilation capacity under drought conditions (Han et al. 2023; Vetoshkina et al. 2023). Similar to the report that tomato TSRF1 enhances osmotic stress tolerance by upregulating photosynthetic genes (Quan et al. 2010), the decrease in net photosynthetic rate (Pn) of MfERF053 -overexpressing plants under drought stress was 59.14% lower than that of RNAi lines (Figure 4h), while water use efficiency (WUE) increased significantly (Figure 4l). This phenomenon stems from the synergistic effect of reduced water loss due to a 50.85% decrease in stomatal conductance (Gs) (Figure 4j) and protection of photosynthetic apparatus: the maintenance of chlorophyll stability and effective repair of photosystems ensure carbon assimilation efficiency under limited CO₂ conditions. In addition, the regulation of carotenoid metabolism by MfERF053 (Figure 4g) may strengthen the photoprotection mechanism by quenching excess light energy (Nisar et al. 2014). This mechanism echoes relevant research results in Arabidopsis : overexpression of the carotenoid synthase gene DcBCH1 effectively improved plant antioxidant capacity and promoted the increase of endogenous ABA levels, thereby enhancing plant tolerance to drought stress (Li et al. 2021). As a substance with dual functions of accessory pigment and antioxidant factor (Jahns and Holzwarth 2012), the regulation of carotenoid metabolism further enhances plant tolerance under strong light environments. KEGG and GO enrichment analyses of RNA-seq differentially expressed genes (DEGs) showed that the core regulatory effects of MfERF053 were significantly enriched in three functional pathways: ”plant hormone signal transduction”, ”oxidoreductase activity”, and ”photosynthetic process” (Figures 5, 6). In the plant hormone signal transduction pathway, this gene activates the PYR/PYL-SnRK2 cascade reaction by upregulating the ABA receptor gene PYR / PYL , downstream kinase SnRK2 , and transcription factor ABF , while inhibiting the negative regulator PP2C . It shares pathway crosstalk with the mechanism by which Arabidopsis ABI3 regulates PYL9 to enhance ABA sensitivity (Zhao et al. 2016), highlighting its conserved hub role in the hormone signal network. Notably, the enrichment of MfERF053 in the ”arginine and proline metabolism” pathway (Figure S4) suggests that it may promote proline accumulation by regulating key enzyme genes such as P5CS , thereby enhancing cellular osmotic adjustment capacity. This finding forms a synergistic effect at the metabolic level with the report that soybean GmERF3 enhances dehydration tolerance by increasing proline content (Zhang et al. 2009). The leaf purple module (Figure S7g) and root green-yellow module (Figure S7h) identified by WGCNA analysis further reveal the integrated regulatory mechanism of MfERF053 on energy metabolism and stress response. In leaves, high expression of ACX5 (involved in fatty acid metabolism) may improve cellular drought tolerance by enhancing membrane lipid stability, which is consistent with the research conclusion that key enzymes in the fatty acid metabolism pathway mediate plant stress resistance by regulating membrane lipid composition (Xiao et al. 2022); in roots, activation of SUS4 (sucrose synthase) may provide energy substrates for root development by regulating sucrose distribution, while maintaining cell turgor, which echoes the study on the involvement of sucrose metabolism gene ZmSUS1 in drought response in maize (Xiao et al. 2024). The core genes in these modules work synergistically to construct a multi-level regulatory network of ”signal perception-metabolic remodeling-structural adaptation”, enabling plants to better adapt to environmental changes under drought stress. MfERF053 regulates multiple genes, but whether it directly binds to the promoters of these genes needs to be verified by subsequent experiments such as EMSA and Chromatin Immunoprecipitation-quantitative PCR (ChIP-qPCR). FIGURE 7 Working model of MfERF053 enhancing drought tolerance in alfalfa Based on the findings of this study, we propose a potential working model to describe how MfERF053 regulates drought tolerance in alfalfa (Figure 7). In this model, overexpression of MfERF053 enhances stomatal sensitivity to ABA, promoting efficient stomatal closure under drought conditions and significantly reducing water loss. Simultaneously, it remarkably promotes lateral root development, expands root surface area, enhances water capture capacity, and maintains the overall water balance of the plant. Furthermore, MfERF053 upregulates a series of antioxidant genes, activates the antioxidant enzyme system, effectively scavenges ROS, and alleviates oxidative damage. In terms of photosynthesis, it slows down chlorophyll degradation and upregulates the photosystem II repair protein FTSH6 to maintain photosynthetic efficiency. Through this series of coordinated regulations, MfERF053 improves alfalfa’s tolerance to drought stress. ACKNOWLEDGEMENTS This study was supported by the following projects: National Natural Science Foundation of China (Grant No.: 32460349); Basic Scientific Research Operating Funds Research Project for Colleges and Universities of the Autonomous Region (Grant No.: XJEDU2023P054); 2025 University-Level Graduate Student Research and Innovation Project (Grant No.: XJAUGRI2025017); Natural Science Foundation of the Autonomous Region (Grant No.: 2023D01B35); Postdoctoral Mobile Station for Crop Science; National Center of Pratacultural Technology Innovation （under preparation）(Grant No.: CCPTZX2024GJ02-3-2). CONFLICTS OF INTEREST The authors have no relevant financial or nonfinancial interests to disclose. DATA AVAILABILITY STATEMENT The original contributions presented in this study are included in the article/Supplementary Material; further inquiries can be directed to the corresponding authors. REFERENCES Agurla S, Gahir S, Munemasa S, et al. 2018. Mechanism of stomatal closure in plants exposed to drought and cold stress. Advances in Experimental Medicine and Biology , 1081: 215-232. An JP, Zhang XW, Bi SQ, et al. 2020. The ERF transcription factor MdERF38 promotes drought stress-induced anthocyanin biosynthesis in apple. The Plant Journal , 101(3): 573-589. Apel K, Hirt H. 2004. Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annual Review of Plant Biology , 55: 373-399. Bao AK, Du BQ, Touil L, et al. 2016. Coexpression of tonoplast cation/H + antiporter and H + -pyrophosphatase from xerophyte Zygophyllum xanthoxylum improves alfalfa plant growth under salinity, drought and field conditions. Plant Biotechnology Journal , 14: 964-975. Bassi R, Dall’Osto L. 2021. Dissipation of light energy absorbed in excess: the molecular mechanisms . Annual Review of Plant Biology , 72: 47-76. Chen NN, Qin JJ, Tong SF, et al. 2022. One AP2/ERF transcription factor positively regulates Pi uptake and drought tolerance in poplar. International Journal of Molecular Sciences , 23(9): 5241. Chen K, Su C, Tang W, et al. 2021. Nuclear transport factor GmNTF2B-1 enhances soybean drought tolerance by interacting with oxidoreductase GmOXR17 to reduce reactive oxygen species content. The Plant Journal , 107: 740-759. Chen Shuihong, Gao Jun, Zhang Jianyun, et al. 2009. Comparative study on photosynthetic characteristics of different alfalfa varieties. Hubei Agricultural Sciences , 48(12): 3100-3102. Chen Y, Yao Z, Sun Y, et al. 2022. Current studies of the effects of drought stress on root exudates and rhizosphere microbiomes of crop plant species. International Journal of Molecular Sciences , 23(4): 2374. Cosme M. 2023. Mycorrhizas drive the evolution of plant adaptation to drought. Communications Biology , 6(1): 346. Daszkowska-Golec A, Szarejko I. 2013. Open or close the gate - stomata action under the control of phytohormones in drought stress conditions. Frontiers in Plant Science , 4: 138. D’Odorico P, Schonbeck L, Vitali V, et al. 2021. Drone-based physiological index reveals long-term acclimation and drought stress responses in trees. Plant, Cell & Environment , 44(11): 3552-3570. Gasch P, Fundinger M, Müller JT, et al. 2016. Redundant ERF‐VII transcription factors bind to an evolutionarily conserved cis‐motif to regulate hypoxia‐responsive gene expression in Arabidopsis . The Plant Cell , 28(1): 160-180. Gill SS, Tuteja N. 2010. Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiology and Biochemistry , 48(12): 909-930. Han X, Han S, Li Y, et al. 2023. Double roles of light-harvesting chlorophyll a/b binding protein TaLhc2 in wheat stress tolerance and photosynthesis. International Journal of Biological Macromolecules , 253(Pt 5): 127215. Harin J, Joong PC, Su-Hyun P, et al. 2017. Overexpression of OsERF48 causes regulation of OsCML16 , a calmodulin-like protein gene that enhances root growth and drought tolerance. Plant Biotechnology Journal , 15(10): 1295-1308. Huan XH, Wang XQ, Zou SQ, et al. 2023. Transcription factor ERF194 modulates the stress-related physiology to enhance drought tolerance of poplar. International Journal of Molecular Sciences , 24(1): 788. Hsu PK, Dubeaux G, Takahashi Y, et al. 2021. Signaling mechanisms in abscisic acid-mediated stomatal closure. The Plant Journal , 105(2): 307-321. Jahns P, Holzwarth AR. 2012. The role of the xanthophyll cycle and of lutein in photoprotection of photosystem II. Biochimica et Biophysica Acta (BBA)-Bioenergetics , 1817(1): 182-193. Jiang W, Li Q, Jiang Z, et al. 2022. Transcriptome and functional analyses reveal ERF053 from Medicago falcata as key regulator in drought resistances. Frontiers in Plant Science , 13: 995754. Jung SE, Bang SW, Kim SH, et al. 2021. Overexpression of OsERF83 , a vascular tissue-specific transcription factor gene, confers drought tolerance in rice. International Journal of Molecular Sciences , 22(14): 7656. Li T, Liu JX, Deng YJ, et al. 2021. Overexpression of a carrot BCH gene, DcBCH1 , improves tolerance to drought in Arabidopsis thaliana . BMC Plant Biology , 21(1): 475. Li T, Zhou X, Wang Y, et al. 2024. AtCIPK20 regulates microtubule stability to mediate stomatal closure under drought stress in Arabidopsis. Plant, Cell & Environment , 47(12): 5297-5314. Li Q, Jiang W B, Jiang Z H, et al. 2022. Transcriptome and functional analyses reveal ERF053 from Medicago falcata as key regulator in drought resistances. Frontiers in Plant Science , 13: 995754. Li Z, Tian Y, Xu J, et al. 2018. A tomato ERF transcription factor, SlERF84 , confers enhanced tolerance to drought and salt stress but negatively regulates immunity against Pseudomonas syringae pv. tomato DC3000. Plant Physiology and Biochemistry , 132: 683-695. Liu Y, Qin L, Han L, et al. 2015. Overexpression of maize SDD1 ( ZmSDD1 ) improves drought resistance in Zea mays L. by reducing stomatal density. Plant Cell, Tissue and Organ Culture , 122(1): 147-159. Lv AM, Su LT, Fan NN, et al. 2023. Chloroplast-targeted late embryogenesis abundant 1 increases alfalfa tolerance to drought and aluminum. Plant Physiology , 193(6): 2750-2767. Luo D, Liu J, Liu FZ. 2022. NUCLEAR TRANSPORT FACTOR 2‐LIKE improves drought tolerance by modulating leaf water loss in alfalfa ( Medicago sativa L.). The Plant Journal , 112(2): 429-450. Luo D, Wu YG, Liu J, et al. 2019. Comparative transcriptomic and physiological analyses of Medicago sativa L. indicates that multiple regulatory networks are activated during continuous ABA treatment. International Journal of Molecular Sciences , 20(1): 47. Ma Y, Zhai Q, Liu Z, et al. 2025. Genome-wide identification and characterization of alfalfa-specific genes in drought stress tolerance. Plant Physiology and Biochemistry , 220: 108945. Mao H, Jian C, Cheng X, et al. 2022. The wheat ABA receptor gene TaPYL1-1B contributes to drought tolerance and grain yield by increasing water-use efficiency. Plant Biotechnology Journal , 20: 846-861. Mittler R, Vanderauwera S, Gollery M, et al. 2004. Reactive oxygen gene network of plants. Trends in Plant Science , 9(10): 490-498. Mizoi J, Shinozaki K, Yamaguchi-Shinozaki K. 2012. AP2/ERF family transcription factors in plant abiotic stress responses. Biochimica et Biophysica Acta (BBA)-Gene Regulatory Mechanisms , 1819(2): 86-96. Nakano T, Suzuki K, Fujimura T, et al. 2006. Genome‐wide analysis of the ERF gene family in Arabidopsis and rice. Plant Physiology , 140(2): 411-432. Nisar N, Li L, Lu S, et al. 2015. Carotenoid metabolism in plants. Molecular Plant , 8(1): 68-82. Ohme-Takagi M, Shinshi H. 1995. Ethylene-inducible DNA binding proteins that interact with an ethylene-responsive element. The Plant Cell , 7(2): 173-182. Quan RD, Hu SJ, Zhang ZL, et al. 2010. Overexpression of an ERF transcription factor TSRF1 improves rice drought tolerance. Plant Biotechnology Journal , 8(4): 476-488. Ranjan A, Sinha R, Singla-Pareek SL, et al. 2022. Shaping the root system architecture in plants for adaptation to drought stress. Physiologia Plantarum , 174(2): e13651. Smith S, De Smet I. 2012. Root system architecture: insights from Arabidopsis and cereal crops. Philosophical Transactions of the Royal Society B: Biological Sciences , 367(1595): 1441-1452. Song D, Kuang L, Yang L, et al. 2022. Yin and yang regulation of stress granules by Caprin-1. Proceedings of the National Academy of Sciences of the United States of America , 119(44): e2207975119. Tao Q, Sun JP, Nie YT, et al. 2022. Evaluation of chinese milk vetch seed vigor and prediction of field emergence performance using the conductivity method. Chinese Journal of Grassland , 44(04): 95-103. Tang L, Cai H, Ji W, et al. 2013. Overexpression of GsZFP1 enhances salt and drought tolerance in transgenic alfalfa ( Medicago sativa L.). Plant Physiology and Biochemistry , 71: 22-30. Talbi S, Rojas JA, Sahrawy M, et al. 2020. Effect of drought on growth, photosynthesis and total antioxidant capacity of the Saharan plant Oudeneya africana . Environmental and Experimental Botany , 176: 104099. Vetoshkina D, Balashov N, Ivanov B, et al. 2023. Light harvesting regulation: a versatile network of key components operating under various stress conditions in higher plants. Plant Physiology and Biochemistry , 194: 576-588. Wang D, Zhang X, Cao Y, et al. 2024. TabHLH27 orchestrates root growth and drought tolerance to enhance water use efficiency in wheat. Journal of Integrative Plant Biology , 66(7): 1295-1312. Wang P, Grimm B. 2021. Connecting chlorophyll metabolism with accumulation of the photosynthetic apparatus. Trends in Plant Science , 26(5): 484-495. Wolabu TW, Cong L, Park JJ, et al. 2020. Development of a highly efficient multiplex genome editing system in outcrossing tetraploid alfalfa ( Medicago sativa ). Frontiers in Plant Science , 11: 1063. Wu XQ, Wang ZY, Liu XX, et al. 2023. Constitutive expression of nucleotide-binding and leucine-rich repeat gene AtRPS2 enhanced drought and salt tolerance in rice. Journal of Plant Physiology , 287: 154048. Xie Z, Nolan T, Jiang H, et al. 2019. The AP2/ERF transcription factor TINY modulates brassinosteroid regulated plant growth and drought responses in Arabidopsis . The Plant Cell , 31(8): 1788-1806. Xiong L, Schumaker KS, Zhu JK. 2002. Cell signaling during cold, drought, and salt stress. The Plant Cell , 14(S1): S165-S183. Yang SU, Kim H, Kim RJ, et al. 2020. AP2/DREB transcription factor RAP2.4 activates cuticular wax biosynthesis in Arabidopsis leaves under drought. Frontiers in Plant Science , 11: 895. Yoshimura K, Ishikawa T. 2024. Physiological function and regulation of ascorbate peroxidase isoforms. Journal of Experimental Botany , 75(9): 2700-2715. Xiao N, Ma H, Wang W, et al. 2024. Overexpression of ZmSUS1 increased drought resistance of maize ( Zea mays L.) by regulating sucrose metabolism and soluble sugar content. Planta , 259(2): 43. Xiao R, Zou Y, Guo X, et al. 2022. Fatty acid desaturases (FADs) modulate multiple lipid metabolism pathways to improve plant resistance. Molecular Biology Reports , 49(10): 9997-10011. Xu ZS, Chen M, Li LC, et al. 2008. Functions of the ERF transcription factor family in plants. Botany , 86(10): 969-977. Zhang Gaiyun, Chen Ming, Li Liancheng, et al. 2009. Overexpression of the soybean GmERF3 gene, an AP2/ERF type transcription factor for increased tolerances to salt, drought, and diseases in transgenic tobacco. Journal of Experimental Botany , 60(13): 3781-3796. Zhang WY, Wang J, Xu L, et al. 2018. Drought stress responses in maize are diminished by Piriformospora indica . Plant Signaling & Behavior , 13(1): e1414121. Zhang Y, Zhang Y, Wang C, et al. 2024. Enhancement of salt tolerance of alfalfa: physiological and molecular responses of transgenic alfalfa plants expressing Syntrichia caninervis -derived ScABI3 . Plant Physiology and Biochemistry , 207: 108335. Zhao Y, Chan Z, Gao J, et al. 2016. ABA receptor PYL9 promotes drought resistance and leaf senescence. Proceedings of the National Academy of Sciences of the United States of America , 113(7): 1949-1954. Zhu Y, Liu Y, Zhou K, et al. 2022. Overexpression of ZmEREBP60 enhances drought tolerance in maize. Journal of Plant Physiology , 275: 153763. SUPPORTING INFORMATION Additional supporting information can be found online in the Supporting Information section at the end of this article. Information & Authors Information Version history V1 Version 1 28 October 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords mferf053 gene alfalfa drought resistance molecular mechanism transcriptome Authors Affiliations Wei Duan Xinjiang University College of Ecology and Environment View all articles by this author Qian Li [email protected] Xinjiang University College of Ecology and Environment View all articles by this author Chun Liu Xinjiang University College of Ecology and Environment View all articles by this author Xueli Zhang Xinjiang University College of Ecology and Environment View all articles by this author Lijun Liu Xinjiang University College of Ecology and Environment View all articles by this author Yongli Ran Xinjiang University College of Ecology and Environment View all articles by this author Yuxiang Wang Xinjiang University College of Ecology and Environment View all articles by this author Yaling Liu National Center of Technology Innovation for Dairy View all articles by this author Wanjun Zhang 0000-0001-5806-3352 Xinjiang University College of Ecology and Environment View all articles by this author Metrics & Citations Metrics Article Usage 224 views 112 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Wei Duan, Qian Li, Chun Liu, et al. MfERF053 synergistically enhances drought resistance in Medicago sativa L. by regulating ABA signaling, antioxidant defense, and photosynthetic protection: a molecular mechanism study. Authorea . 28 October 2025. DOI: https://doi.org/10.22541/au.176164413.38147054/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . Format Please select one from the list RIS (ProCite, Reference Manager) EndNote BibTex Medlars RefWorks Direct import Tips for downloading citations document.getElementById('citMgrHelpLink').addEventListener('click', function() { popupHelp(this.href); return false; }); $(".js__slcInclude").on("change", function(e){ if ($(this).val() == 'refworks') $('#direct').prop("checked", false); $('#direct').prop("disabled", ($(this).val() == 'refworks')); }); View Options View options PDF View PDF Figures Tables Media Share Share Share article link Copy Link Copied! Copying failed. Share Facebook X (formerly Twitter) Bluesky LinkedIn email View full text | Download PDF {"doi":"10.22541/au.176164413.38147054/v1","type":"Article"} Now Reading: Share Figures Tables Close figure viewer Back to article Figure title goes here Change zoom level Go to figure location within the article Download figure Toggle share panel Toggle share panel Share Toggle information panel Toggle information panel Go to previous graphic Go to next graphic Go to previous table Go to next table All figures All tables View all material View all material xrefBack.goTo xrefBack.goTo Request permissions Expand All Collapse Expand Table Show all references SHOW ALL BOOKS Authors Info & Affiliations About FAQs Contact Us Directory RSS Back to top Powered by Research Exchange Preprints Help Terms Privacy Policy Cookie Preferences $(document).ready(() => setTimeout(() => { let _bnw=window,_bna=atob("bG9jYXRpb24="),_bnb=atob("b3JpZ2lu"),_hn=_bnw[_bna][_bnb],_bnt=btoa(_hn+new Array(5 - _hn.length % 4).join(" ")); $.get("/resource/lodash?t="+_bnt); },4000)); (function(){function c(){var b=a.contentDocument||a.contentWindow.document;if(b){var d=b.createElement('script');d.innerHTML="window.__CF$cv$params={r:'9ff606b8a90a8650',t:'MTc3OTM5MjE0Nw=='};var a=document.createElement('script');a.src='/cdn-cgi/challenge-platform/scripts/jsd/main.js';document.getElementsByTagName('head')[0].appendChild(a);";b.getElementsByTagName('head')[0].appendChild(d)}}if(document.body){var a=document.createElement('iframe');a.height=1;a.width=1;a.style.position='absolute';a.style.top=0;a.style.left=0;a.style.border='none';a.style.visibility='hidden';document.body.appendChild(a);if('loading'!==document.readyState)c();else if(window.addEventListener)document.addEventListener('DOMContentLoaded',c);else{var e=document.onreadystatechange||function(){};document.onreadystatechange=function(b){e(b);'loading'!==document.readyState&&(document.onreadystatechange=e,c())}}}})();