AhDTF1, a novel R2R3-MYB transcription factor, involves in drought tolerance and seed color

AhDTF1, a novel R2R3-MYB transcription factor, involves in drought tolerance and seed color | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article AhDTF1, a novel R2R3-MYB transcription factor, involves in drought tolerance and seed color Sijian Wang, Yiwen Yang, Jianbiao Huang, Honglin Zhao, Shuaixia Wu, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7702056/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 13 You are reading this latest preprint version Abstract Background Peanut ( Arachis hypogaea L.), a major oilseed crop globally, suffers significant yield reduction due to drought stress. Here, we identified a novel R2R3-MYB transcription factor AhDTF1 through phylogenetic analysis, comprehensive transcriptomic analysis of drought responses, and tissue expression pattern. Result AhDTF1 functioned as a nuclear transcriptional activator and positively regulates drought resistance. Transgenic tobacco plants over-expressing AhDTF1 exhibited significantly enhanced drought tolerance, manifested by elevated activities of antioxidant enzymes such as POD, SOD, APX, CAT, and GST, increased soluble protein content, and reduced MDA accumulation compared to WT. Furthermore, AhDTF1 activated the expression of key stress-responsive genes, including APX , CAT , POD , SOD , GST , CAX3 , and OAT . AhDTF1 also regulated seed coat color and root hair development under abiotic stress conditions, Hap3 conferring longer root length. These findings demonstrated that AhDTF1 played a critical role in plant drought stress adaptation by coordinating antioxidant defense mechanisms and osmotic adjustment. Our work highlighted AhDTF1 as a promising candidate for crop improvement, providing a dual-tolerance strategy against drought and associated oxidative stresses through MYB-dependent transcriptional regulation. peanut R2R3-MYB AhDTF1 transcriptional activator drought tolerance antioxidant Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Drought is one of the most common abiotic stresses that plants encounter in natural environments, and it severely limits crop productivity [ 1 ]. Severe drought stress causes morphological impairment, disruption of cellular structures, and dysregulation of metabolic processes [ 2 – 5 ]. To cope with water deficit, plants use a range of regulated physiological and molecular mechanisms to reduce water loss, sustain water supply to critical tissues, maintain cellular hydration, and activate antioxidant systems for cellular protection [ 1 ]. For instance, plants modulate stomatal aperture to reduce transpiration, a process strongly influenced by the phytohormone ABA, which triggers stomatal closure via guard cell contraction [ 6 – 8 ]. Additionally, adaptive modifications in root architecture—such as increased root length and density—improve water uptake capacity in dry soils [ 9 – 11 ]. Plant cells also accumulate osmoprotectants like proline and compatible solutes to stabilize osmotic potential and cellular water status [ 12 ]. Concurrently, antioxidant enzymes, including SOD, POD, and GST, are activated to remove ROS and reduce oxidative damage [ 13 – 16 ]. Plants have evolved complex regulatory networks to respond to drought stress, with TFs playing central roles. Key TF families involved include AP2/ERF, bHLH, bZIP, DREB, GATA, MADS-box, MYB, NAC, and WRKY [ 17 , 18 ]. ABA is a major regulator in drought responses, mediating both ABA-dependent and ABA-independent transcriptional pathways [ 19 – 22 ]. These TFs bind to ABREs to control downstream gene expression [ 23 , 24 ] and take part in stress signal perception, transduction, and adaptive responses [ 18 , 25 ]. By activating or repressing specific target genes, they affect various processes such as stomatal movement, oxidative stress mitigation, and leaf senescence [ 26 , 27 ]. MYB transcription factors represent one of the largest TF families in plants. Since their first discovery in maize, they have been widely studied. MYB proteins are defined by one to four imperfect amino acid repeats (approximately 52 residues each), which form DNA-binding domains. Each repeat consists of three α-helices, with the second and third forming a HTH structure stabilized by three conserved tryptophan residues [ 28 ]. Based on the number of repeats, MYB proteins are divided into four subclasses, among which R2R3-MYB is the most common in plants [ 29 , 30 ]. Progress in molecular biology has allowed better functional analysis of MYB TFs, highlighting their importance in regulating drought responses [ 31 , 32 ]. Specific members, such as MYB60, are involved in controlling stomatal movement and drought adaptation [ 33 , 34 ]. Others, including MYB41, MYB94, and MYB96, control the synthesis of cuticular waxes, which are important for reducing water loss and improving drought tolerance [ 35 – 39 ]. Moreover, certain MYB TFs take part in flavonoid biosynthesis, providing antioxidant protection under drought and oxidative stress [ 40 , 41 ]. Some MYB factors also directly or indirectly affect the expression of antioxidant enzyme genes, helping remove ROS and maintain redox homeostasis during drought [ 13 , 15 , 42 ]. Despite these advances, studies on MYB TFs in peanut under drought conditions are still limited. Liu et al. [ 43 ] reported differential expression of six AhMYB44 genes under drought stress, with tests in Arabidopsis showing that AhMYB44-11 and AhMYB44-16 act as positive and negative regulators of drought tolerance, respectively. Recent transcriptomic and metabolomic studies have provided more details on the physiological and molecular mechanisms behind drought adaptation in peanut, including antioxidant defense, photosynthetic adjustment, osmotic regulation, and activation of signaling pathways [ 32 , 42 , 44 – 47 ]. These insights are useful for finding drought-responsive genes in peanut. In this study, we identified an R2R3-MYB transcription factor, AhDTF1, through phylogenetic analysis and integration of drought stress transcriptomic data. Transgenic Arabidopsis thaliana and Nicotiana tabacum plants over-expressing AhDTF1 showed better drought tolerance under water-deficient conditions. Tests revealed that AhDTF1 affects root system architecture and increases the antioxidant defense system and Hap3 conferring longer root length. Our results indicate that AhDTF1 is a good candidate gene for improving drought tolerance in crops through transcriptional regulation. Results Identification of the drought tolerance factor AhDTF1 We found 246 AhR2R3-MYB genes in this study, which is 50 more than those in our previous analysis, due to improved genome annotation [ 48 ] (Additional file 1). Sequence alignment confirmed that these genes contain typical R2R3-MYB domains (Additional file 2). We built a phylogenetic tree using the 246 AhR2R3-MYB proteins and 11 drought-related AtMYB proteins (AtMYB12, AtMYB15, AtMYB20, AtMYB37, AtMYB41, AtMYB60, AtMYB75, AtMYB88, AtMYB94, AtMYB96, and AtMYB124) [ 32 ] (Additional file 3, Additional file 4). Based on tree structure and bootstrap values (> 75), five drought-associated clades containing 32 AhR2R3-MYBs (Fig. 1 A) were identified. To find more drought-responsive R2R3-MYB genes, we used two drought-induced transcriptome datasets [ 44 , 47 ]. Only one orthologous gene pair ( Ah02g107800 and Ah12g133000 ) was found in both the drought-responsive R2R3-MYBs and the drought-related phylogenetic clades (Fig. 1 B). Ah02g107800 and Ah12g133000 , located on chromosomes 2 and 12, have similar gene structures with three exons and conserved intron phases (Fig. 1 C). The proteins are 280 and 284 amino acids long, with identical N-terminal domains but small differences at their C-termini (Fig. 1 D). Although both genes had similar expression patterns across 22 peanut tissues, Ah12g133000 had higher expression levels and was chosen as the candidate drought tolerance gene for further study (Fig. 1 E). AhDTF1 is a nucleus-located transcription activator To find where AhDTF1 is located in the cell, we expressed an EGFP-AhDTF1 fusion construct in tobacco epidermal cells. Confocal microscopy showed that the EGFP-AhDTF1 signal was only in the nucleus and matched the nuclear marker AtHistone H1.2-TagRFP [ 49 ] (Fig. 2 A). To test if AhDTF1 can activate transcription, we used a yeast-based assay. Yeast cells expressing AhDTF1 grew normally and turned blue on selective medium (SD/-Trp-Leu with AbA and X-α-gal). We made a series of shortened versions of AhDTF1 (ΔN1-194, ΔN1-284, and ΔC195-284) and found that only the C-terminal part (amino acids 195–284) kept strong transactivation activity, shown by good growth and blue staining (Fig. 2 B). These results show that AhDTF1 is a nuclear-localized transcriptional activator, with its transactivation domain in the C-terminal region (195–284 aa). AhDTF1 positively regulates drought stress response To test the role of AhDTF1 in drought stress tolerance, we made stable over-expression lines in Arabidopsis and tobacco, and did transient over-expression in peanut. After checking transgenic integration by PCR, we picked three independent lines for functional tests (Additional file 5). RT-qPCR proved high expression of AhDTF1 in these lines (Additional file 5). We did drought treatment and recovery tests with AhDTF1 -overexpressing and WT Arabidopsis plants. After 14 days without water, WT plants wilted more than the over-expression lines. After 7 days of rewatering, the over-expression lines had much higher survival rates than WT (Fig. 3 A, B). Under normal conditions, AhDTF1 -overexpressing and WT tobacco seeds germinated at similar rates (Fig. 4 A, B). However, with higher mannitol concentrations, germination rates dropped in both types, but transgenic lines kept better germination under all stress levels. No major differences in fresh weight, root length, or root-shoot ratio were seen between WT and transgenic seedlings under normal conditions (Fig. 5 A, B). Under mannitol stress, WT plants had less biomass as concentration increased, while transgenic lines kept relatively stable growth. At 100 mM mannitol, transgenic plants had 12.5% higher fresh weight than WT (Fig. 5 A). Microscopy showed a 3-fold rise in root hair density in transgenic plants under 100 mM mannitol compared to WT (Additional file 6). Although root length decreased with higher mannitol in both types, transgenic plants had longer roots at 200 mM (42% longer) and 300 mM (58% longer) (Fig. 5 B). The higher root hair density and better root elongation led to a steady higher root-shoot ratio (27% greater) in transgenic plants under stress. Also, AhDTF1 -overexpressing tobacco seeds had lighter seed coat color than WT (Additional file 7). Transient over-expression of AhDTF1 in peanut also improved drought resistance compared to WT plants (Additional file 8). We identified three nonsynonymous SNP variants in the promotor region of AhDTF1 that have MAF > 0.05. Haplotype analysis revealed that three major haplotypes formed by these three SNPs were associated with the difference in root length between CK and drought stress treatment and Hap3 conferring longer root length. We performed promoter sequencing analysis of the three haplotypes of AhDTF1 , Hap3 leads to a new TAAT box and the disappearance of a DNA methylation site (Fig. 6 ). Over-expression of AhDTF1 raises SOD and POD activities Drought stress turns on antioxidant systems to keep cellular redox balance. To measure physiological changes, we tested the POD and SOD activities, and the levels of soluble proteins and MDA. Under normal conditions, AhDTF1 -overexpressing plants had higher POD and SOD activities than WT, and these activities went up further under drought stress (Fig. 7 A, B). MDA contents were similar between types under control conditions but much lower in transgenic lines under drought (Fig. 7 C). Soluble protein contents were higher in transgenic lines L12, L23, and L29 under control conditions. At 100 mM mannitol, all overexpression lines had more soluble proteins than WT (Fig. 7 D). Over expression of AhDTF1 increases oxidative stress-related genes To understand the molecular mechanisms behind better drought tolerance, we checked the expression of antioxidant and drought-responsive genes in transgenic tobacco. Under normal conditions, the expression levels of APX , CAT , CAX3 , SOD , GST , DREB3 , and NCED1 were similar between transgenic and WT plants (Fig. 8 A-I). Under drought stress, ROS-scavenging genes were much higher in transgenic plants, matching the rise in POD and SOD activities. In contrast, the expression of DREB3 and NCED1 was lower. Discussion Finding candidate genes through transcriptome difference analysis is a common method [ 50 ]. However, results can change with experimental conditions like cultivar, tissue type, and treatment time, making it hard to find key regulators only from transcriptomics. Although many drought-related transcriptomic studies have been done in peanut, most give general views rather than functional details on single genes [ 32 , 44 , 42 , 45 – 47 ]. Since members of the same phylogenetic group often have similar functions, comparing with known genes can help predict function [ 51 ]. So, combining drought-responsive transcriptomics with phylogenetic analysis is a good way to find drought tolerance genes. The functional tests of AhDTF1 here show this method works. Because both Ah02g107800 and AhDTF1 were found through mixed phylogenetic and transcriptomic analysis, we think Ah02g107800 may also have a role in abiotic stress responses in peanut. Hap3 of AhDTF1 leads to a new TAAT box and the disappearance of a DNA methylation site, which results in differences in its gene expression, with upregulation of OsDTF1 expression promoting root elongation under drought conditions, thereby enhancing drought resistance. In the drought-associated group, AtMYB15 is the Arabidopsis protein closest to AhDTF1, but with low sequence similarity (Additional file 9). AtMYB15 does not have clear tissue-specific expression (Additional file 10), while both AhDTF1 and Ah02g107800 are mainly expressed in underground tissues (e.g., developing pods, roots, and gynophores) and have lower expression in above-ground parts like leaves (Fig. 1 E). This suggests that AhDTF1 may be a new type of drought-responsive regulator. Root growth in soil must overcome physical limits like compaction and resistance. High expression in root tissues may therefore show abiotic stress resistance. For example, the potato PYL16 gene, which is highly expressed in roots, gives drought resistance [ 52 ]. Since genes often work where they are expressed [ 53 ], the better root growth—shown by higher root-shoot ratio and root hair density—seen in AhDTF1 -overexpressing plants under mannitol stress fits with its expression pattern. AhDTF1 seems to give two benefits: promoting root growth under stress and boosting cellular antioxidant ability (Fig. 9 ). The lighter seed coat color in transgenic materials may reflect more antioxidant activity. As a geocarpic species, peanut has fertilization above ground but fruit development below ground. The high expression of Ah02g107800 and AhDTF1 in below-ground tissues suggests important roles in pod development. Peanut is a major global oil and cash crop. However, its low reproduction rate and complex allotetraploid genome (AABB, 2n = 4x = 40) make genetic mapping hard with standard linkage analysis. Even large association studies have difficulty measuring complex traits like drought tolerance. This study shows how combining evolutionary-phylogenetic analysis, transcriptomic profiling, and expression checks can quickly find stress-related regulators. With screening of natural variants, this method can help find better alleles for breeding stress-resistant cultivars. Our work provides a method for finding genes in peanut and genetic material for drought tolerance breeding. Conclusions This study describes a novel peanut R2R3-MYB transcription factor, AhDTF1, which functions as a nuclear transcriptional activator that positively regulates drought resistance. Transgenic tobacco overexpressing AhDTF1 exhibited significantly enhanced drought tolerance, characterized by elevated activities of antioxidant enzymes (POD, SOD, APX, CAT, GST), increased soluble protein content, and reduced malondialdehyde (MDA) accumulation, indicating mitigated oxidative damage. AhDTF1 activates the expression of key stress-responsive genes, including those encoding antioxidant enzymes and other critical factors such as CAX3 and OAT, thereby coordinating antioxidant defense and osmotic adjustment. Furthermore, AhDTF1 regulates seed coat color and root hair development under abiotic stress, with the Hap3 haplotype associated with longer root length, enhancing stress adaptation. In conclusion, AhDTF1 plays a critical role in plant drought adaptation through MYB-dependent transcriptional regulation of antioxidant and osmotic processes. It provides a dual-tolerance mechanism against drought and oxidative stress, making it a promising candidate gene for improving crop resilience in breeding programs. Materials and methods Plant growth and stress treatments Seeds of peanut variety 'Changhua 18' were kept in our laboratory. After soaking in water for 4 h, seeds were sown in a 3:1 (v/v) mix of nutrient soil and vermiculite and grown in a greenhouse at 28°C with a 16-h light/8-h dark period. For Arabidopsis thaliana drought treatments, seeds were sown in the same soil mix and grown at 22°C under 16-h light/8-h dark cycles. After four weeks, water was stopped for 14 days then resumed. For tobacco tests, seeds were sown on full MS medium or MS medium with 100, 200, or 300 mM mannitol. Tobacco plants were kept at 25°C under 16-h light/8-dark conditions.The cultivation of all the aforementioned materials was conducted in the greenhouse at Jiangxi Agricultural University, Nanchang City, Jiangxi Province, China. Sequence and phylogenetic analysis AhR2R3-MYB sequences were re-checked using the latest peanut genome annotation (Tifrunner.gnm2) as in Wang et al. [ 48 ]. Eleven drought-related AtR2R3-MYB sequences were taken from TAIR [ 32 ]. Multiple sequence alignment was done with MAFFT v7.520 [ 54 ]. Phylogenetic analysis used the Neighbor-Joining method with the JTT model and 1000 bootstrap repeats. Gene annotations came from PeanutBase [ 55 ]. Expression Analysis Peanut expression data were downloaded from PeanutBase [ 55 ]. Drought-stress transcriptome data were from previous studies [ 44 , 47 ]. Difference expression data are in Additional file 11. AtMYB15 expression data were from TAIR. Affymetrix ATH1 array data were normalized with the GCOS method and a TGT value of 100 [ 56 ]. For tobacco gene expression tests, 20-day-old plants were split into control and treatment (100 mM mannitol for 24 h) groups. Leaf tissues were collected, frozen in liquid nitrogen, and stored at − 80°C. Total RNA was taken out using RNAiso Plus (Takara Bio Inc.). cDNA was made with the PrimeScript™ FAST RT reagent Kit with gDNA Eraser (Takara Bio Inc.). qRT-PCR was done on an ABI 7500 system (Applied Biosystems, USA) with TB Green® Premix Ex Taq™ II (Tli RNaseH Plus) under these steps: 95°C for 30 s, then 40 cycles of 95°C for 5 s and 60°C for 34 s. Each reaction was done three times, and relative expression was found with the 2 − ΔΔCt method. Gene-specific primers are in Additional file 12. Subcellular localization The coding sequence of AhDTF1 was amplified and put into the pCAMBIA1300-EGFP vector to make a 35S:EGFP-AhDTF1 fusion construct. The nuclear marker AtHistone H1.2-TagRFP [ 49 ] was used as a co-transfection control. Constructs were put into tobacco epidermal cells through agroinfiltration and kept at 25°C for 36–48 h. GFP and RFP signals were seen with a confocal laser-scanning microscope (Olympus FV4000, Japan). Primers are in Additional file 13. Transcriptional activation assay The full-length CDS of AhDTF1 and its shortened parts (N-terminal: 1–161 aa; middle: 143–221 aa; C-terminal: 195–284 aa) were cloned into the pGBKT7 vector. Each construct was put with pGADT7 into Y2H Gold yeast cells and placed on SD/-Trp-Leu and SD/-Trp-Leu/X-α-gal/AbA media. Yeast transformation and growth followed the Clontech manual. Primers are in Table S5. Vectorconstructs and plant transformation The full-length CDS of AhDTF1 was cloned into the pQB-V3 vector (with attL1/attL2 sites) and then moved to pGWB511 via Gateway™ LR Clonase™ II recombination (Invitrogen, Cat. #11791020). Nicotiana tabacum leaf discs were transformed with Agrobacterium tumefaciens strain EHA105. Arabidopsis thaliana (Col-0) was transformed by floral dip with A. tumefaciens strain GV3101. Transgenic plants were selected on hygromycin B and checked by PCR. T2 generation lines were used for tests. For transient overexpression in peanut, a bacterial suspension (GV3101 with 35S:AhDTF1) was put into the lower leaf surfaces of variety 'Changhua 06'. After 72 h in the dark, plants were treated with drought using 20% PEG. Plants were grown in water at 25°C for 14 days before treatment. Measurement of soluble protein and MDA content Soluble protein content was found with the Bradford method [ 57 ] with BSA as standard. For MDA content, leaf samples (0.5 g) were ground in 0.6% thiobarbituric acid and heated in a boiling water bath for 15 min. After centrifugation at 12,000 × g for 15 min, absorbance was read at 450 nm, 532 nm, and 600 nm [ 58 ]. Antioxidant enzyme activity tests Enzyme extraction was done as in Sekmen et al. [ 59 ] with changes. Leaf tissues (0.5 g) were ground in 10 mL phosphate buffer (PBS, pH 7.8) and spun at 10,000 rpm for 15 min at 4°C. The supernatant was the enzyme extract. SOD activity was measured by how it stopped nitroblue tetrazolium (NBT) photoreduction [ 60 ]. The reaction mix had 130 mM PBS (pH 7.8), 130 mM methionine, 750 µM NBT, 100 µM EDTA, 20 µM riboflavin, and 20 µL enzyme extract. After 30 min light (4000 lux), absorbance was read at 560 nm. POD activity was tested with guaiacol as substrate [61]. One unit of POD activity was set as an increase of 0.01 in absorbance per minute at 470 nm. Abbreviations AhDTF1 Drought Tolerance Factor 1 POD Peroxidase SOD Superoxide dismutase APX ascorbate peroxidase, CAT:Catalase GST Glutathione S-transferase MDA malondialdehyde WT wild-type CAX3 vacuolar cation/proton exchanger 3 and OAT (ornithine aminotransferase). ABA abscisic acid ROS reactive oxygen TFs transcription factors ABREs ABA-responsive elements HTH helix-turn-helix Declarations Author's Contributions LYW, HCM, and JHF designed the project. SJW,YWY, and JBH performed data analysis. SJW, YWY, SXW, JBH and YL conducted the experiments. SJW wrote the original manuscript. LYW and HCM contributed to the interpretation of data. LYW and HCM revised the manuscript. Acknowledgments We thank Prof. Juncheng Zhang from Hangzhou Normal University, for supplying the vectors. Funding This work was supported by Jiangxi Province Key R&D Program Project (20243BBH81028), the the Jiangxi Agriculture Research System (No.JCARS-02), Special Topics on Sustainable Agricultural Science and Technology Innovation of Xinjiang Academy of Agricultural Sciences (xjnkywdzc-2023001-27), and the National Natural Science Foundation of China (32160433 and 32160101). Data availability Data is provided within the manuscript or supplementary information files. Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests References Gupta A, Rico-Medina A, Caño-Delgado AI. The physiology of plant responses to drought. Science. 2020;368:266–9. Nakashima K, Tran LS, Van Nguyen D, Fujita M, Maruyama K, et al. Functional analysis of a NAC-type transcription factor OsNAC6 involved in abiotic and biotic stress-responsive gene expression in rice. Plant J. 2007;51:617–30. Choudhury FK, Rivero RM, Blumwald E, Mittler R. Reactive oxygen species, abiotic stress and stress combination. Plant J. 2017;90:856–67. Tardieu F, Simonneau T, Muller B. The physiological basis of drought tolerance in crop plants: a scenario-dependent probabilistic approach. Annu Rev Plant Biol. 2018;69:733–59. Ha Y, Shang Y, Nam KH. Brassinosteroids modulate ABA-induced stomatal closure in Arabidopsis . J Exp Bot. 2016;67:6297–308. Brodribb TJ, McAdam SA. Evolution of the stomatal regulation of plant water content. Plant Physiol. 2017;174:639–49. Batool S, Uslu VV, Rajab H, Ahmad N, Waadt R, et al. Sulfate is incorporated into cysteine to trigger ABA production and stomatal closure. Plant Cell. 2018;30:2973–87. Rellan-Alvarez R, Lobet G, Dinneny JR. Environmental control of root system biology. Annu Rev Plant Biol. 2016;67:619–42. Dinneny JR. Developmental responses to water and salinity in root systems. Annu Rev Cell Dev Biol. 2019;35:239–57. Ranjan A, Sinha R, Singla-Pareek SL, Pareek A, Singh AK. Shaping the root system architecture in plants for adaptation to drought stress. Physiol Plant. 2022;174:e13651. Fabregas N, Lozano-Elena F, Blasco-Escámez D, Tohge T, Martínez-Andújar C, et al. Overexpression of the vascular brassinosteroid receptor BRL3 confers drought resistance without penalizing plant growth. Nat Commun. 2018;9:4680. Chen T, Li W, Hu X, Guo J, Liu A, et al. A cotton MYB transcription factor, GbMYB5, is positively involved in plant adaptive response to drought stress. Plant Cell Physiol. 2015;56:917–29. Wang N, Zhang W, Qin M, Li S, Qiao M, et al. Drought tolerance conferred in soybean ( Glycine max . L) by GmMYB84, a novel R2R3-MYB transcription factor. Plant Cell Physiol. 2017;58:1764–76. Bailey-Serres J, Parker JE, Ainsworth EA, Oldroyd GED, Schroeder JI. Genetic strategies for improving crop yields. Nature. 2019;575:109–18. Cui X, Tang M, Li L, Chang J, Yang X, et al. Expression patterns and molecular mechanisms regulating drought tolerance of soybean [ Glycine max (L.) Merr.] conferred by transcription factor gene GmNAC19 . Int J Mol Sci. 2024;25:1234. Cominelli E, Galbiati M, Tonelli C. Transcription factors controlling stomatal movements and drought tolerance. Transcription. 2010;1:41–5. Hussain Q, Asim M, Zhang R, Khan R, Farooq S, et al. Transcription factors interact with ABA through gene expression and signaling pathways to mitigate drought and salinity stress. Biomolecules. 2021;11:1159. Shinozaki K, Yamaguchi-Shinozaki K. Molecular responses to dehydration and low temperature: differences and cross-talk between two stress signaling pathways. Curr Opin Plant Biol. 2000;3:217–23. Yamaguchi-Shinozaki K, Shinozaki K. Organization of cis-acting regulatory elements in osmotic- and cold-stress-responsive promoters. Trends Plant Sci. 2005;10:88–94. Ahn TS, Ka JO, Lee GH, Song HG. Microcosm study for revegetation of barren land with wild plants by some plant growth-promoting rhizobacteria. J Microbiol Biotechnol. 2007;17:52–7. Cutler SR, Rodriguez PL, Finkelstein RR, Abrams SR. Abscisic acid: emergence of a core signaling network. Annu Rev Plant Biol. 2010;61:651–79. Tuteja N. Abscisic acid and abiotic stress signaling. Plant Signal Behav. 2007;2:135–8. Yang W, Liu XD, Chi XJ, Wu CA, Li YZ, et al. Dwarf apple MbDREB1 enhances plant tolerance to low temperature, drought, and salt stress via both ABA-dependent and ABA-independent pathways. Planta. 2011;233:219–29. Joshi R, Wani SH, Singh B, Bohra A, Dar ZA, et al. Transcription factors and plants response to drought stress: current understanding and future directions. Front Plant Sci. 2016;7:1029. Hu W, Huang C, Deng X, Zhou S, Chen L, et al. TaASR1, a transcription factor gene in wheat, confers drought stress tolerance in transgenic tobacco. Plant Cell Environ. 2013;36:1449–64. Khan SA, Li MZ, Wang SM, Yin HJ. Revisiting the role of plant transcription factors in the battle against abiotic stress. Int J Mol Sci. 2018;19:1634. Ogata K, Kanei-Ishii C, Sasaki M, Hatanaka H, Nagadoi A, et al. The cavity in the hydrophobic core of MYB DNA-binding domain is reserved for DNA recognition and trans-activation. Nat Struct Biol. 1996;3:178–87. Dubos C, Stracke R, Grotewold E, Weisshaar B, Martin C, et al. MYB transcription factors in Arabidopsis . Trends Plant Sci. 2010;15:573–81. Liu J, Osbourn A, Ma P. MYB transcription factors as regulators of phenylpropanoid metabolism in plants. Mol Plant. 2015;8:689–708. Li J, Han G, Sun C, Sui N. Research advances of MYB transcription factors in plant stress resistance and breeding. Plant Signal Behav. 2019;14:1613131. Wang X, Niu Y, Zheng Y. Multiple functions of MYB transcription factors in abiotic stress responses. Int J Mol Sci. 2021;22:6125. Cominelli E, Galbiati M, Vavasseur A, Conti L, Sala T, et al. A guard-cell-specific MYB transcription factor regulates stomatal movements and plant drought tolerance. Curr Biol. 2005;15:1196–200. Oh JE, Kwon Y, Kim JH, Noh H, Hong SW, et al. A dual role for MYB60 in stomatal regulation and root growth of Arabidopsis thaliana under drought stress. Plant Mol Biol. 2011;77:91–103. Cominelli E, Sala T, Calvi D, Gusmaroli G, Tonelli C. Over-expression of the Arabidopsis AtMYB41 gene alters cell expansion and leaf surface permeability. Plant J. 2008;53:53–64. Seo PJ, Lee SB, Suh MC, Park MJ, Go YS, et al. The MYB96 transcription factor regulates cuticular wax biosynthesis under drought conditions in Arabidopsis . Plant Cell. 2011;23:1138–52. Lee SB, Kim H, Kim RJ, Suh MC. Overexpression of Arabidopsis MYB96 confers drought resistance in Camelina sativa via cuticular wax accumulation. Plant Cell Rep. 2014;33:1535–46. Lee SB, Suh MC. Cuticular wax biosynthesis is up-regulated by the MYB94 transcription factor in Arabidopsis . Plant Cell Physiol. 2015;56:48–60. Lee SB, Kim HU, Suh MC. MYB94 and MYB96 additively activate cuticular wax biosynthesis in Arabidopsis . Plant Cell Physiol. 2016;57:2300–11. Nakabayashi R, Yonekura-Sakakibara K, Urano K, Suzuki M, Yamada Y, et al. Enhancement of oxidative and drought tolerance in Arabidopsis by overaccumulation of antioxidant flavonoids. Plant J. 2014;77:367–79. Wang F, Kong W, Wong G, Fu L, Peng R, et al. AtMYB12 regulates flavonoids accumulation and abiotic stress tolerance in transgenic Arabidopsis thaliana . Mol Genet Genomics. 2016;291:1545–59. Zhang P, Wang R, Yang X, Ju Q, Li W, et al. The R2R3-MYB transcription factor AtMYB49 modulates salt tolerance in Arabidopsi s by modulating the cuticle formation and antioxidant defence. Plant Cell Environ. 2020;43:1925–43. Liu Y, Shen Y, Liang M, Zhang X, Xu J, et al. Identification of peanut AhMYB44 transcription factors and their multiple roles in drought stress responses. Plants (Basel). 2022;11:300. Brasileiro ACM, Morgante CV, Araujo ACG, Leal-Bertioli SCM, Silva AK, et al. Transcriptome profiling of wild Arachis from water-limited environments uncovers drought tolerance candidate genes. Plant Mol Biol Rep. 2015;33:1876–92. Zhao N, Cui S, Li X, Liu B, Deng H, et al. Transcriptome and co-expression network analyses reveal differential gene expression and pathways in response to severe drought stress in peanut ( Arachis hypogaea L). Front Genet. 2021;12:672884. Ren J, Guo P, Zhang H, Shi X, Ai X, et al. Comparative physiological and coexpression network analyses reveal the potential drought tolerance mechanism of peanut. BMC Plant Biol. 2022;22:460. Liu X, Su L, Li L, Zhang Z, Li X, et al. Transcriptome profiling reveals characteristics of hairy root and the role of AhGLK1 in response to drought stress and post-drought recovery in peanut. BMC Genomics. 2023;24:119. Wang S, Xu Z, Yang Y, Ren W, Fang J, et al. Genome-wide analysis of R2R3-MYB genes in cultivated peanut ( Arachis hypogaea L.): Gene duplications, functional conservation, and diversification. Front Plant Sci. 2023;14:1102174. Xu J, Lee YJ, Liu B. Establishment of a mitotic model system by transient expression of the D-type cyclin in differentiated leaf cells of tobacco ( Nicotiana benthamiana ). New Phytol. 2020;226:1213–20. Jiang Z, Zhou X, Li R, Michal JJ, Zhang S, et al. Whole transcriptome analysis with sequencing: methods, challenges and potential solutions. Cell Mol Life Sci. 2015;72:3425–39. Jiang C, Gu X, Peterson T. Identification of conserved gene structures and carboxy-terminal motifs in the MYB gene family of Arabidopsis and Oryza sativa L. ssp. indica. Genome Biol. 2004;5:R46. Yao P, Zhang C, Bi Z, Liu Y, Liu Z, Wei J, Su X, Bai J, Cui J, Sun C. Overexpression of potato PYL16 gene in tobacco enhances the transgenic plant tolerance to drought stress. Int J Mol Sci. 2024;25:8644. Lawson MJ, Church GM. Tissue-specific gene expression: Mechanisms and evolutionary implications. Nat Rev Genet. 2022;22:305–22. Katoh K, Rozewicki J, Yamada KD. MAFFT online service: multiple sequence alignment, interactive sequence choice and visualization. Brief Bioinform. 2019;20:1160–6. Clevenger J, Chu Y, Scheffler B, Ozias-Akins P. A developmental transcriptome map for allotetraploid Arachis hypogaea . Front Plant Sci. 2016;7:644. Schmid M, Davison TS, Henz SR, Pape UJ, Demar M, et al. A gene expression map of Arabidopsis thaliana development. Nat Genet. 2005;37:501–6. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248–54. Heath RL, Packer L. Reprint of: Photoperoxidation in isolated chloroplasts. I. Kinetics and stoichiometry of fatty acid peroxidation. Arch Biochem Biophys. 2022;726:109248. Sekmen AH, Ozgur R, Uzilday B, Turkan I. Reactive oxygen species scavenging capacities of cotton ( Gossypium hirsutum ) cultivars under combined drought and heat induced oxidative stress. Environ Exp Bot. 2014;99:141–9. Giannopolitis CN, Ries SK. Superoxide dismutases: II. Purification and quantitative relationship with water-soluble protein in seedlings. Plant Physiol. 1977;59:315–8. Miao BH, Han XG, Zhang WH. The ameliorative effect of silicon on soybean seedlings grown in potassium-deficient medium. Ann Bot. 2010;105:967–73. Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterial.rar SupplementaryinformationLegend.docx Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 15 May, 2026 Reviews received at journal 06 May, 2026 Reviews received at journal 03 May, 2026 Reviewers agreed at journal 27 Apr, 2026 Reviewers agreed at journal 23 Apr, 2026 Reviews received at journal 24 Feb, 2026 Reviewers agreed at journal 27 Jan, 2026 Reviewers agreed at journal 27 Dec, 2025 Reviewers invited by journal 25 Dec, 2025 Editor invited by journal 18 Dec, 2025 Editor assigned by journal 14 Oct, 2025 Submission checks completed at journal 14 Oct, 2025 First submitted to journal 14 Oct, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7702056","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":566203196,"identity":"48a5a315-fba4-4810-80fd-93fa19877fd7","order_by":0,"name":"Sijian Wang","email":"","orcid":"","institution":"JiangxiAgricultural University","correspondingAuthor":false,"prefix":"","firstName":"Sijian","middleName":"","lastName":"Wang","suffix":""},{"id":566203197,"identity":"efab49c8-792d-4b75-b5ca-7b19e34fef33","order_by":1,"name":"Yiwen Yang","email":"","orcid":"","institution":"JiangxiAgricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yiwen","middleName":"","lastName":"Yang","suffix":""},{"id":566203198,"identity":"8f3e414c-c125-4d85-ad6f-66d3ae9bfbda","order_by":2,"name":"Jianbiao Huang","email":"","orcid":"","institution":"JiangxiAgricultural University","correspondingAuthor":false,"prefix":"","firstName":"Jianbiao","middleName":"","lastName":"Huang","suffix":""},{"id":566203199,"identity":"1a64e745-b00c-444f-a39a-20dabafe0bdf","order_by":3,"name":"Honglin Zhao","email":"","orcid":"","institution":"JiangxiAgricultural University","correspondingAuthor":false,"prefix":"","firstName":"Honglin","middleName":"","lastName":"Zhao","suffix":""},{"id":566203200,"identity":"e2d4009d-2180-4282-87ec-719c0865e80f","order_by":4,"name":"Shuaixia Wu","email":"","orcid":"","institution":"JiangxiAgricultural University","correspondingAuthor":false,"prefix":"","firstName":"Shuaixia","middleName":"","lastName":"Wu","suffix":""},{"id":566203201,"identity":"8f1b5cf1-a30d-48eb-a7bf-f8acb35273eb","order_by":5,"name":"Shuwei Liu","email":"","orcid":"","institution":"JiangxiAgricultural University","correspondingAuthor":false,"prefix":"","firstName":"Shuwei","middleName":"","lastName":"Liu","suffix":""},{"id":566203205,"identity":"72aa69db-cb82-4f18-8f43-edaf8cd89a87","order_by":6,"name":"Yao Lu","email":"","orcid":"","institution":"JiangxiAgricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yao","middleName":"","lastName":"Lu","suffix":""},{"id":566203206,"identity":"d5269523-6a2c-4e49-b4d9-7952217d914e","order_by":7,"name":"Weifang Ren","email":"","orcid":"","institution":"JiangxiAgricultural University","correspondingAuthor":false,"prefix":"","firstName":"Weifang","middleName":"","lastName":"Ren","suffix":""},{"id":566203207,"identity":"4cc35c01-1003-4fa1-a282-54e25c99c627","order_by":8,"name":"Xincheng Xu","email":"","orcid":"","institution":"JiangxiAgricultural University","correspondingAuthor":false,"prefix":"","firstName":"Xincheng","middleName":"","lastName":"Xu","suffix":""},{"id":566203208,"identity":"42690fd1-6dcd-4923-bea1-d40a19665f18","order_by":9,"name":"Jiahai Fang","email":"","orcid":"","institution":"JiangxiAgricultural University","correspondingAuthor":false,"prefix":"","firstName":"Jiahai","middleName":"","lastName":"Fang","suffix":""},{"id":566203210,"identity":"e0bef8d6-e174-41ef-bd71-af62523ed8fa","order_by":10,"name":"Haocui Miao","email":"","orcid":"","institution":"Crop Research Institute of Xinjiang Uygur Autonomous Region Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Haocui","middleName":"","lastName":"Miao","suffix":""},{"id":566203211,"identity":"ac67185b-0e8e-433c-bc30-4bf11df54aea","order_by":11,"name":"Liyun Wan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA50lEQVRIiWNgGAWjYHACxgMJQJKfmbHxwQcDGzui9IC1SLY3NxvOKEhLJk4LiDA4c7xNmufDIcYGQsoNjp89cOBh2+E8hhuJbdI2BgeYGdgPH92AV8uZvIQDiW2HixlnJDZb5xjc4WPgSUu7gU+L2YEcA5CWxGaJxMbbOQbPmBkkeMzwazn/BqKlTSKxQdrC4DBjA0EtN6C29PAcbJJmIEaL/Q2gLQnn0hNnsDc2G/YYpCWzEfKLZH+O4cMfZdaJ+w+zP3zw44+NHT/74WN4tYABIxsShw2nMhTwhzhlo2AUjIJRMEIBAFw6VlWt5lLcAAAAAElFTkSuQmCC","orcid":"","institution":"JiangxiAgricultural University","correspondingAuthor":true,"prefix":"","firstName":"Liyun","middleName":"","lastName":"Wan","suffix":""}],"badges":[],"createdAt":"2025-09-24 09:38:44","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7702056/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7702056/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":99314603,"identity":"14fd1f79-3f78-4c2d-ae24-83e588942d33","added_by":"auto","created_at":"2025-12-31 16:22:00","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":121315,"visible":true,"origin":"","legend":"","description":"","filename":"AhDTF1MS20251014BMCP.docx","url":"https://assets-eu.researchsquare.com/files/rs-7702056/v1/5291cd6656db88e6f26d0faf.docx"},{"id":99128354,"identity":"b84992d5-0e3b-4d61-b4dd-ba65fcfb1690","added_by":"auto","created_at":"2025-12-29 03:40:52","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":6334065,"visible":true,"origin":"","legend":"","description":"","filename":"Figure11.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7702056/v1/ec1e99b41a5cb77f860919c1.jpg"},{"id":99128345,"identity":"e47b4d28-bfde-4a74-a41d-e1b6eab946bd","added_by":"auto","created_at":"2025-12-29 03:40:51","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":934519,"visible":true,"origin":"","legend":"","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7702056/v1/bd0598c4c1c7dd8c51977791.jpg"},{"id":99316101,"identity":"627bfadc-0859-4fe5-8477-2909e1218748","added_by":"auto","created_at":"2025-12-31 16:27:44","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":4842173,"visible":true,"origin":"","legend":"","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7702056/v1/8410e02056a7a7a3232a5e94.jpg"},{"id":99315463,"identity":"9c811819-d2e8-4c0c-84d0-f6170e8aad46","added_by":"auto","created_at":"2025-12-31 16:26:56","extension":"jpg","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1309968,"visible":true,"origin":"","legend":"","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7702056/v1/c7d11d0325d649d454f7e275.jpg"},{"id":99315643,"identity":"5e758185-1588-4d8d-a444-d2b3715d7274","added_by":"auto","created_at":"2025-12-31 16:27:11","extension":"jpg","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":8684092,"visible":true,"origin":"","legend":"","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7702056/v1/df13503b887bf5f1a3531e36.jpg"},{"id":99128347,"identity":"61ef56e5-02e8-4845-a6dc-198176603ef9","added_by":"auto","created_at":"2025-12-29 03:40:52","extension":"jpg","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":219927,"visible":true,"origin":"","legend":"","description":"","filename":"Figure6n.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7702056/v1/300ca65cc83cd91a061fa7b9.jpg"},{"id":99128369,"identity":"b0006926-3574-4cfc-9e07-ffa186899173","added_by":"auto","created_at":"2025-12-29 03:40:52","extension":"jpg","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":525980,"visible":true,"origin":"","legend":"","description":"","filename":"Figure72.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7702056/v1/3bd73b2b00152fc5f9bd5ffd.jpg"},{"id":99128371,"identity":"dd8c2559-6a81-44eb-a70b-50103d934f28","added_by":"auto","created_at":"2025-12-29 03:40:52","extension":"jpg","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1206100,"visible":true,"origin":"","legend":"","description":"","filename":"Figure8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7702056/v1/d7e48c0ae24e48160d6bb9a9.jpg"},{"id":99128353,"identity":"0d28e8a1-3e61-4591-8163-734192616990","added_by":"auto","created_at":"2025-12-29 03:40:52","extension":"jpg","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":184928,"visible":true,"origin":"","legend":"","description":"","filename":"Figure9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7702056/v1/aa2f4dd6a8071fa3f6b0650a.jpg"},{"id":99314948,"identity":"39f21b8c-fe32-4b24-aa11-40b7ba81e4bf","added_by":"auto","created_at":"2025-12-31 16:25:05","extension":"json","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":11528,"visible":true,"origin":"","legend":"","description":"","filename":"38249926ba794b85bd9f924153ed64d8.json","url":"https://assets-eu.researchsquare.com/files/rs-7702056/v1/12e922ac8400c1e58d09e6d1.json"},{"id":99128358,"identity":"98a0221d-5778-4985-888d-edea17854a41","added_by":"auto","created_at":"2025-12-29 03:40:52","extension":"rar","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":42024593,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.rar","url":"https://assets-eu.researchsquare.com/files/rs-7702056/v1/ea17511c2a79190fab4d5c49.rar"},{"id":99128364,"identity":"d7aa1d21-8341-4160-8c2c-5bd5a8f1ded8","added_by":"auto","created_at":"2025-12-29 03:40:52","extension":"xml","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":130175,"visible":true,"origin":"","legend":"","description":"","filename":"38249926ba794b85bd9f924153ed64d81enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-7702056/v1/627c994af6cbec199f9a161b.xml"},{"id":99128368,"identity":"30eceb3a-d56b-4532-ba3d-a56fb4151a1c","added_by":"auto","created_at":"2025-12-29 03:40:52","extension":"jpg","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":6334065,"visible":true,"origin":"","legend":"","description":"","filename":"Figure11.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7702056/v1/71ab4bf6df79e2dee9b890b1.jpg"},{"id":99128363,"identity":"01f7874a-01af-49f5-85ac-3602cb675c76","added_by":"auto","created_at":"2025-12-29 03:40:52","extension":"jpg","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":934519,"visible":true,"origin":"","legend":"","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7702056/v1/9d9ad17d42dc26322ad282f6.jpg"},{"id":99128380,"identity":"187a1965-08f2-4570-91ee-da9fa31b2956","added_by":"auto","created_at":"2025-12-29 03:40:53","extension":"jpg","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":4842173,"visible":true,"origin":"","legend":"","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7702056/v1/507594c77092a5fdee9bf5ce.jpg"},{"id":99128365,"identity":"fe30bef1-b494-4b1f-bae8-71e5624c4f6f","added_by":"auto","created_at":"2025-12-29 03:40:52","extension":"jpg","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1309968,"visible":true,"origin":"","legend":"","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7702056/v1/8c8e83ecd46dd24495844198.jpg"},{"id":99128357,"identity":"019e31ba-ec38-4bd8-99c5-cc99e3aacc14","added_by":"auto","created_at":"2025-12-29 03:40:52","extension":"jpg","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":8684092,"visible":true,"origin":"","legend":"","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7702056/v1/d126c03ea021615e66609b32.jpg"},{"id":99314893,"identity":"b96bca02-ce38-4a0a-9117-697923090f64","added_by":"auto","created_at":"2025-12-31 16:24:21","extension":"jpg","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":219927,"visible":true,"origin":"","legend":"","description":"","filename":"Figure6n.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7702056/v1/f68c434ffa85ac94ea594b71.jpg"},{"id":99128375,"identity":"12b2c2b2-c19c-4539-9804-e17f073e4e1a","added_by":"auto","created_at":"2025-12-29 03:40:52","extension":"jpg","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":525980,"visible":true,"origin":"","legend":"","description":"","filename":"Figure72.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7702056/v1/ec15eb462cb05b0d67680f67.jpg"},{"id":99128385,"identity":"4021b76b-54e4-4b3c-b1ae-499138f42604","added_by":"auto","created_at":"2025-12-29 03:40:53","extension":"jpg","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1206100,"visible":true,"origin":"","legend":"","description":"","filename":"Figure8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7702056/v1/d292a7cbd7365b0e2755f871.jpg"},{"id":99128367,"identity":"eefda7f8-c25a-4305-83c2-e2d8b7ba7a47","added_by":"auto","created_at":"2025-12-29 03:40:52","extension":"jpg","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":184928,"visible":true,"origin":"","legend":"","description":"","filename":"Figure9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7702056/v1/607d1054a318c15a951abfed.jpg"},{"id":99316461,"identity":"b58d647b-5974-41fa-badb-fba1409f89e8","added_by":"auto","created_at":"2025-12-31 16:28:29","extension":"png","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2807006,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure11.png","url":"https://assets-eu.researchsquare.com/files/rs-7702056/v1/cac07ce20a2562e2601e5c88.png"},{"id":99128373,"identity":"a4582b2e-2b9d-43d9-89de-727ef65c7b3a","added_by":"auto","created_at":"2025-12-29 03:40:52","extension":"png","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":206943,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7702056/v1/79d9d54e8262da8f2e56be22.png"},{"id":99314602,"identity":"6f32923d-d298-48e3-9c10-860d8eb7538b","added_by":"auto","created_at":"2025-12-31 16:21:59","extension":"png","order_by":24,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2108562,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7702056/v1/c6e75e8eab046a87bfde3141.png"},{"id":99128370,"identity":"b039d5ae-b2f8-4b6b-8874-3355a79b2362","added_by":"auto","created_at":"2025-12-29 03:40:52","extension":"png","order_by":25,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":881915,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7702056/v1/a92275b0ec9d81884973bb91.png"},{"id":99315254,"identity":"7c01d009-cbaa-413e-bab1-83ab83ac9814","added_by":"auto","created_at":"2025-12-31 16:26:42","extension":"png","order_by":26,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2478552,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7702056/v1/c8c426a2885917bfbd86a881.png"},{"id":99314696,"identity":"2bdeee75-cfc3-4b8b-b6d8-c200e8291867","added_by":"auto","created_at":"2025-12-31 16:22:22","extension":"png","order_by":27,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":90208,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure6n.png","url":"https://assets-eu.researchsquare.com/files/rs-7702056/v1/322a6a88e16585680c50fdbb.png"},{"id":99315071,"identity":"bad2440a-4725-4a01-983b-f545685fba82","added_by":"auto","created_at":"2025-12-31 16:26:17","extension":"png","order_by":28,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":146497,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure72.png","url":"https://assets-eu.researchsquare.com/files/rs-7702056/v1/83ac44bace262a9266b5a715.png"},{"id":99128377,"identity":"42edd2e5-ecbc-4a34-a806-eb4b06b8417b","added_by":"auto","created_at":"2025-12-29 03:40:52","extension":"png","order_by":29,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":356564,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure8.png","url":"https://assets-eu.researchsquare.com/files/rs-7702056/v1/a7578cb27761dcd8d52c5829.png"},{"id":99315070,"identity":"ff4449d3-39c8-467c-98db-fb00de5e43fe","added_by":"auto","created_at":"2025-12-31 16:26:17","extension":"png","order_by":30,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":116470,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure9.png","url":"https://assets-eu.researchsquare.com/files/rs-7702056/v1/c4c6a436ad8281c3ca0a0944.png"},{"id":99315373,"identity":"ff923846-ed4f-4177-972e-0e2c4626b906","added_by":"auto","created_at":"2025-12-31 16:26:52","extension":"xml","order_by":31,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":128648,"visible":true,"origin":"","legend":"","description":"","filename":"38249926ba794b85bd9f924153ed64d81structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7702056/v1/e8d7f4cd7a90be0a1d29a575.xml"},{"id":99128382,"identity":"8d9b3480-f395-4388-ad9c-5b0f178146cd","added_by":"auto","created_at":"2025-12-29 03:40:53","extension":"html","order_by":32,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":144596,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7702056/v1/4cdc573c22a2fc7d2601bbac.html"},{"id":99128344,"identity":"b3115cd0-c559-4cde-bbba-b425ebb00fc1","added_by":"auto","created_at":"2025-12-29 03:40:51","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":6334065,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSequence and expression analysis of\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e AhDTF1.\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003ePhylogenetic tree of the 246 AhR2R3-MYBand the 11 drought-responsive AtR2R3-MYB proteins. Collapsed branches indicated by black circles (see Figure S2). (B) Venn diagram of three drought-related gene datasets: Ad \u0026amp; As (differentially expressed genes under drought stress in \u003cem\u003eArachis duranensis \u003c/em\u003eand \u003cem\u003eA. stenosperma\u003c/em\u003e), Homologous (AhR2R3-MYBs highly homologous to \u003cem\u003eArabidopsis thaliana \u003c/em\u003edrought-responsive genes), and HR (differentially expressed genes under drought/recovery in peanut hairy root systems). (C) Gene structures of \u003cem\u003eAh12g133000 \u003c/em\u003e(\u003cem\u003eAhDTF1\u003c/em\u003e) and \u003cem\u003eAh02g107800\u003c/em\u003e. (D) Protein sequence alignment of Ah12g133000 (AhDTF1) and Ah02g107800. (E) Tissue-specific expression profiles of \u003cem\u003eAh12g133000 \u003c/em\u003eand \u003cem\u003eAh02g107800 \u003c/em\u003eacross 22 peanut tissues. The meanings of the abbreviations of the 22 tissues were as follows: seedling leaf 10 days post-emergence (leaf 1), main stem leaf (leaf 2), lateral stem leaf (leaf 3), vegetative shoot tip from the main stem (veg shoot), reproductive shoot tip from first lateral (repr shoot), 10-day roots (root), 25-day nodules (nodule), perianth, stamen, pistil, aerial gynophore tip (peg tip 1), subterranean peg tip (peg tip 2), Pattee 1 stalk (peg tip Pat. 1), Pattee 1 pod (fruit Pat. 1), Pattee 3 pod (fruit Pat. 3), Pattee 5 pericarp (pericarp Pat. 5), Pattee 6 pericarp (pericarp Pat. 6), Pat - tee 5 seed (seed Pat. 5), Pattee 6 seed (seed Pat. 6), Pattee 7 seed (seed Pat. 7), Pattee 8 seed (seed Pat. 8), and Pattee 10 seed (seed Pat. 10). TPM values (standardized) as the gene expression levels (*P \u0026lt; 0.05 and **P \u0026lt; 0.01; t-test).\u003c/p\u003e","description":"","filename":"Figure11.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7702056/v1/ad3ea99a14a33b2cab7ab94b.jpg"},{"id":99128349,"identity":"77126cf1-5873-488d-9903-448b8745a2f5","added_by":"auto","created_at":"2025-12-29 03:40:52","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":934519,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterization of the transcription factor AhDTF1.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Subcellular localization of AhDTF1 in tobacco leaves. Bars = 30 μm (B) Transcriptional activation analysis of AhDTF1 in yeast. The transcriptional activation ability was analyzed by growth on DDO (SD/-Trp -Leu ) and DDO/A/X (SD/-Trp-Leu with Aureobasidin A and X-α-gal) plates.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7702056/v1/ae7e05d65f62320a251ce4ff.jpg"},{"id":99315753,"identity":"2c8d64c7-d874-4641-87f6-3184b1e3c07c","added_by":"auto","created_at":"2025-12-31 16:27:19","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4842173,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIncreased drought tolerance of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAhDTF1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-OE \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eArabidopsis \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eplants.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Phenotype changes of WT and \u003cem\u003eAhDTF1\u003c/em\u003e-OE \u003cem\u003eArabidopsis\u003c/em\u003eplants before/after drought stress. (B) The survival rate of seedlings from (A).\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7702056/v1/4c7b9ce148a63ac61e01d121.jpg"},{"id":99128351,"identity":"b24015b1-b648-4152-a99b-a1e55bb9d105","added_by":"auto","created_at":"2025-12-29 03:40:52","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1309968,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOver-expression of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAhDTF1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e improved germination in tobacco under drought stress. \u003c/strong\u003e(A) Comparison of germination of \u003cem\u003eAhDTF1\u003c/em\u003e transgenic tobacco and wild type at different mannitol concentrations. (B) Germination of tobacco plants over-expressing\u003cem\u003eAhDTF1\u003c/em\u003eand wild type at different mannitol concentrations at 5th, 7th, 11th and 14th day after sowing.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7702056/v1/cdf13972adc50c335947edff.jpg"},{"id":99315608,"identity":"27998acb-26e2-4756-afcc-48ddb3127601","added_by":"auto","created_at":"2025-12-31 16:27:07","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":8684092,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhenotypic changes in tobacco plants over-expressing \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAhDTF1 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eand wild type at different mannitol concentrations.\u003c/strong\u003e (A) Growth of \u003cem\u003eAhDTF1\u003c/em\u003e-OE tobacco and wild type at different mannitol concentrations. Bars = 2cm. (B) Fresh weight, root length and root to crown ratio of \u003cem\u003eAhDTF1\u003c/em\u003e-OE tobacco and wild type at different mannitol concentrations. Asterisks indicate a significant difference between WT and OE lines by t-test, *P \u0026lt; 0.05 and **P \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7702056/v1/9e7d578bf7bfbb8dd3af8ebe.jpg"},{"id":99128356,"identity":"e2953b3f-c5da-405a-b5a1-8fb3f1ba505d","added_by":"auto","created_at":"2025-12-29 03:40:52","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":219927,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHaplotype analysis of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAhDTF1\u003c/strong\u003e\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"Figure6n.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7702056/v1/c2aed3a344f6fdc33c67cc13.jpg"},{"id":99315829,"identity":"80baee72-5e8f-4d35-8d8a-e7605e241679","added_by":"auto","created_at":"2025-12-31 16:27:23","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":525980,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAntioxidant enzyme activities, soluble proteins and MDA contents of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAhDTF1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-OE tobacco and WT under drought stress.\u003c/strong\u003e (A) POD activity in tobacco after 100 mM mannitol for 20 d. (B) SOD activity in tobacco after 100 mM mannitol for 20 d. (C) MDA contents in tobacco treated with 100 mM mannitol for 20 d. (D) Soluble protein contents in tobacco treated with 100 mM mannitol for 20 d. Asterisks indicate a significant difference between WT and OE lines by t-test, *P \u0026lt; 0.05 and **P \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Figure72.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7702056/v1/dca43b371bb7c422028549ef.jpg"},{"id":99316163,"identity":"01219e2a-326a-47ba-bbb6-44098c96462f","added_by":"auto","created_at":"2025-12-31 16:27:48","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1206100,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression patterns of abiotic stress-responsive genes in transgenic and WT tobacco under normal and drought stress conditions.\u003c/strong\u003e (A-I) Expression of \u003cem\u003eAPX\u003c/em\u003e, \u003cem\u003eCAT\u003c/em\u003e, \u003cem\u003eCAX3\u003c/em\u003e, \u003cem\u003ePOD\u003c/em\u003e, \u003cem\u003eSOD\u003c/em\u003e, \u003cem\u003eGST\u003c/em\u003e, \u003cem\u003eOAT\u003c/em\u003e, \u003cem\u003eDREB3,\u003c/em\u003e and \u003cem\u003eNCED1\u003c/em\u003ein WT and \u003cem\u003eAhDTF1\u003c/em\u003e-OE tobacco seedlings under 100 mM mannitol conditions for 20 d. Data are means ± SE of three biological replicates. Statistical significance was calculated using Student’s t test (*P \u0026lt; 0.05 and **P \u0026lt; 0.01). NS, no significant difference.\u003c/p\u003e","description":"","filename":"Figure8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7702056/v1/ce681b6ef69a8f3d410f0fd5.jpg"},{"id":99314823,"identity":"53a80447-9ad0-472a-a278-2e6bd7bc5315","added_by":"auto","created_at":"2025-12-31 16:23:26","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":184928,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHypothetical model of AhDTF1 in drought stress response.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7702056/v1/ce8de8a79e64d1ac9cc6abe4.jpg"},{"id":100406148,"identity":"1f54b4c4-b20f-4800-b71a-461b674f674a","added_by":"auto","created_at":"2026-01-16 12:44:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":25302048,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7702056/v1/b2dbf3cf-f2bc-49cd-8730-3632dd50ff37.pdf"},{"id":99128378,"identity":"aa84b19f-6a06-485a-8673-f62e158efe48","added_by":"auto","created_at":"2025-12-29 03:40:53","extension":"rar","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":42024593,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.rar","url":"https://assets-eu.researchsquare.com/files/rs-7702056/v1/97245d5fd28f042930a969f6.rar"},{"id":99128361,"identity":"9c2e2f04-df1b-409b-ad81-e245ac908481","added_by":"auto","created_at":"2025-12-29 03:40:52","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":15697,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryinformationLegend.docx","url":"https://assets-eu.researchsquare.com/files/rs-7702056/v1/5823941415094f907df4a786.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"AhDTF1, a novel R2R3-MYB transcription factor, involves in drought tolerance and seed color","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDrought is one of the most common abiotic stresses that plants encounter in natural environments, and it severely limits crop productivity [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Severe drought stress causes morphological impairment, disruption of cellular structures, and dysregulation of metabolic processes [\u003cspan additionalcitationids=\"CR3 CR4\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. To cope with water deficit, plants use a range of regulated physiological and molecular mechanisms to reduce water loss, sustain water supply to critical tissues, maintain cellular hydration, and activate antioxidant systems for cellular protection [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. For instance, plants modulate stomatal aperture to reduce transpiration, a process strongly influenced by the phytohormone ABA, which triggers stomatal closure via guard cell contraction [\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Additionally, adaptive modifications in root architecture\u0026mdash;such as increased root length and density\u0026mdash;improve water uptake capacity in dry soils [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Plant cells also accumulate osmoprotectants like proline and compatible solutes to stabilize osmotic potential and cellular water status [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Concurrently, antioxidant enzymes, including SOD, POD, and GST, are activated to remove ROS and reduce oxidative damage [\u003cspan additionalcitationids=\"CR14 CR15\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePlants have evolved complex regulatory networks to respond to drought stress, with TFs playing central roles. Key TF families involved include AP2/ERF, bHLH, bZIP, DREB, GATA, MADS-box, MYB, NAC, and WRKY [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. ABA is a major regulator in drought responses, mediating both ABA-dependent and ABA-independent transcriptional pathways [\u003cspan additionalcitationids=\"CR20 CR21\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. These TFs bind to ABREs to control downstream gene expression [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] and take part in stress signal perception, transduction, and adaptive responses [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. By activating or repressing specific target genes, they affect various processes such as stomatal movement, oxidative stress mitigation, and leaf senescence [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMYB transcription factors represent one of the largest TF families in plants. Since their first discovery in maize, they have been widely studied. MYB proteins are defined by one to four imperfect amino acid repeats (approximately 52 residues each), which form DNA-binding domains. Each repeat consists of three α-helices, with the second and third forming a HTH structure stabilized by three conserved tryptophan residues [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Based on the number of repeats, MYB proteins are divided into four subclasses, among which R2R3-MYB is the most common in plants [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Progress in molecular biology has allowed better functional analysis of MYB TFs, highlighting their importance in regulating drought responses [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Specific members, such as MYB60, are involved in controlling stomatal movement and drought adaptation [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Others, including MYB41, MYB94, and MYB96, control the synthesis of cuticular waxes, which are important for reducing water loss and improving drought tolerance [\u003cspan additionalcitationids=\"CR36 CR37 CR38\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Moreover, certain MYB TFs take part in flavonoid biosynthesis, providing antioxidant protection under drought and oxidative stress [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Some MYB factors also directly or indirectly affect the expression of antioxidant enzyme genes, helping remove ROS and maintain redox homeostasis during drought [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDespite these advances, studies on MYB TFs in peanut under drought conditions are still limited. Liu et al. [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] reported differential expression of six \u003cem\u003eAhMYB44\u003c/em\u003e genes under drought stress, with tests in \u003cem\u003eArabidopsis\u003c/em\u003e showing that \u003cem\u003eAhMYB44-11\u003c/em\u003e and \u003cem\u003eAhMYB44-16\u003c/em\u003e act as positive and negative regulators of drought tolerance, respectively. Recent transcriptomic and metabolomic studies have provided more details on the physiological and molecular mechanisms behind drought adaptation in peanut, including antioxidant defense, photosynthetic adjustment, osmotic regulation, and activation of signaling pathways [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan additionalcitationids=\"CR45 CR46\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. These insights are useful for finding drought-responsive genes in peanut.\u003c/p\u003e \u003cp\u003eIn this study, we identified an R2R3-MYB transcription factor, AhDTF1, through phylogenetic analysis and integration of drought stress transcriptomic data. Transgenic \u003cem\u003eArabidopsis thaliana\u003c/em\u003e and \u003cem\u003eNicotiana tabacum\u003c/em\u003e plants over-expressing \u003cem\u003eAhDTF1\u003c/em\u003e showed better drought tolerance under water-deficient conditions. Tests revealed that AhDTF1 affects root system architecture and increases the antioxidant defense system and Hap3 conferring longer root length. Our results indicate that \u003cem\u003eAhDTF1\u003c/em\u003e is a good candidate gene for improving drought tolerance in crops through transcriptional regulation.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eIdentification of the drought tolerance factor AhDTF1\u003c/h2\u003e \u003cp\u003eWe found 246 AhR2R3-MYB genes in this study, which is 50 more than those in our previous analysis, due to improved genome annotation [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e] (Additional file 1). Sequence alignment confirmed that these genes contain typical R2R3-MYB domains (Additional file 2). We built a phylogenetic tree using the 246 AhR2R3-MYB proteins and 11 drought-related AtMYB proteins (AtMYB12, AtMYB15, AtMYB20, AtMYB37, AtMYB41, AtMYB60, AtMYB75, AtMYB88, AtMYB94, AtMYB96, and AtMYB124) [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] (Additional file 3, Additional file 4). Based on tree structure and bootstrap values (\u0026gt;\u0026thinsp;75), five drought-associated clades containing 32 AhR2R3-MYBs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) were identified. To find more drought-responsive R2R3-MYB genes, we used two drought-induced transcriptome datasets [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Only one orthologous gene pair (\u003cem\u003eAh02g107800\u003c/em\u003e and \u003cem\u003eAh12g133000\u003c/em\u003e) was found in both the drought-responsive R2R3-MYBs and the drought-related phylogenetic clades (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). \u003cem\u003eAh02g107800\u003c/em\u003e and \u003cem\u003eAh12g133000\u003c/em\u003e, located on chromosomes 2 and 12, have similar gene structures with three exons and conserved intron phases (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). The proteins are 280 and 284 amino acids long, with identical N-terminal domains but small differences at their C-termini (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Although both genes had similar expression patterns across 22 peanut tissues, \u003cem\u003eAh12g133000\u003c/em\u003e had higher expression levels and was chosen as the candidate drought tolerance gene for further study (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAhDTF1 is a nucleus-located transcription activator\u003c/h3\u003e\n\u003cp\u003eTo find where AhDTF1 is located in the cell, we expressed an EGFP-AhDTF1 fusion construct in tobacco epidermal cells. Confocal microscopy showed that the EGFP-AhDTF1 signal was only in the nucleus and matched the nuclear marker AtHistone H1.2-TagRFP [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). To test if AhDTF1 can activate transcription, we used a yeast-based assay. Yeast cells expressing AhDTF1 grew normally and turned blue on selective medium (SD/-Trp-Leu with AbA and X-α-gal). We made a series of shortened versions of AhDTF1 (ΔN1-194, ΔN1-284, and ΔC195-284) and found that only the C-terminal part (amino acids 195\u0026ndash;284) kept strong transactivation activity, shown by good growth and blue staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). These results show that AhDTF1 is a nuclear-localized transcriptional activator, with its transactivation domain in the C-terminal region (195\u0026ndash;284 aa).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eAhDTF1 positively regulates drought stress response\u003c/h3\u003e\n\u003cp\u003eTo test the role of AhDTF1 in drought stress tolerance, we made stable over-expression lines in \u003cem\u003eArabidopsis\u003c/em\u003e and tobacco, and did transient over-expression in peanut. After checking transgenic integration by PCR, we picked three independent lines for functional tests (Additional file 5). RT-qPCR proved high expression of \u003cem\u003eAhDTF1\u003c/em\u003e in these lines (Additional file 5).\u003c/p\u003e \u003cp\u003eWe did drought treatment and recovery tests with \u003cem\u003eAhDTF1\u003c/em\u003e-overexpressing and WT \u003cem\u003eArabidopsis\u003c/em\u003e plants. After 14 days without water, WT plants wilted more than the over-expression lines. After 7 days of rewatering, the over-expression lines had much higher survival rates than WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eUnder normal conditions, \u003cem\u003eAhDTF1\u003c/em\u003e-overexpressing and WT tobacco seeds germinated at similar rates (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B). However, with higher mannitol concentrations, germination rates dropped in both types, but transgenic lines kept better germination under all stress levels. No major differences in fresh weight, root length, or root-shoot ratio were seen between WT and transgenic seedlings under normal conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B). Under mannitol stress, WT plants had less biomass as concentration increased, while transgenic lines kept relatively stable growth. At 100 mM mannitol, transgenic plants had 12.5% higher fresh weight than WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Microscopy showed a 3-fold rise in root hair density in transgenic plants under 100 mM mannitol compared to WT (Additional file 6). Although root length decreased with higher mannitol in both types, transgenic plants had longer roots at 200 mM (42% longer) and 300 mM (58% longer) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). The higher root hair density and better root elongation led to a steady higher root-shoot ratio (27% greater) in transgenic plants under stress. Also, \u003cem\u003eAhDTF1\u003c/em\u003e-overexpressing tobacco seeds had lighter seed coat color than WT (Additional file 7). Transient over-expression of \u003cem\u003eAhDTF1\u003c/em\u003e in peanut also improved drought resistance compared to WT plants (Additional file 8).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe identified three nonsynonymous SNP variants in the promotor region of \u003cem\u003eAhDTF1\u003c/em\u003e that have MAF\u0026thinsp;\u0026gt;\u0026thinsp;0.05. Haplotype analysis revealed that three major haplotypes formed by these three SNPs were associated with the difference in root length between CK and drought stress treatment and Hap3 conferring longer root length. We performed promoter sequencing analysis of the three haplotypes of \u003cem\u003eAhDTF1\u003c/em\u003e, Hap3 leads to a new TAAT box and the disappearance of a DNA methylation site (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eOver-expression of\u003c/b\u003e \u003cb\u003eAhDTF1\u003c/b\u003e \u003cb\u003eraises SOD and POD activities\u003c/b\u003e\u003c/p\u003e \u003cp\u003eDrought stress turns on antioxidant systems to keep cellular redox balance. To measure physiological changes, we tested the POD and SOD activities, and the levels of soluble proteins and MDA. Under normal conditions, \u003cem\u003eAhDTF1\u003c/em\u003e-overexpressing plants had higher POD and SOD activities than WT, and these activities went up further under drought stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, B). MDA contents were similar between types under control conditions but much lower in transgenic lines under drought (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). Soluble protein contents were higher in transgenic lines L12, L23, and L29 under control conditions. At 100 mM mannitol, all overexpression lines had more soluble proteins than WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOver expression of AhDTF1 increases oxidative stress-related genes\u003c/p\u003e \u003cp\u003eTo understand the molecular mechanisms behind better drought tolerance, we checked the expression of antioxidant and drought-responsive genes in transgenic tobacco. Under normal conditions, the expression levels of \u003cem\u003eAPX\u003c/em\u003e, \u003cem\u003eCAT\u003c/em\u003e, \u003cem\u003eCAX3\u003c/em\u003e, \u003cem\u003eSOD\u003c/em\u003e, \u003cem\u003eGST\u003c/em\u003e, \u003cem\u003eDREB3\u003c/em\u003e, and \u003cem\u003eNCED1\u003c/em\u003e were similar between transgenic and WT plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA-I). Under drought stress, ROS-scavenging genes were much higher in transgenic plants, matching the rise in POD and SOD activities. In contrast, the expression of \u003cem\u003eDREB3\u003c/em\u003e and \u003cem\u003eNCED1\u003c/em\u003e was lower.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eFinding candidate genes through transcriptome difference analysis is a common method [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. However, results can change with experimental conditions like cultivar, tissue type, and treatment time, making it hard to find key regulators only from transcriptomics. Although many drought-related transcriptomic studies have been done in peanut, most give general views rather than functional details on single genes [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan additionalcitationids=\"CR46\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Since members of the same phylogenetic group often have similar functions, comparing with known genes can help predict function [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. So, combining drought-responsive transcriptomics with phylogenetic analysis is a good way to find drought tolerance genes. The functional tests of \u003cem\u003eAhDTF1\u003c/em\u003e here show this method works. Because both \u003cem\u003eAh02g107800\u003c/em\u003e and \u003cem\u003eAhDTF1\u003c/em\u003e were found through mixed phylogenetic and transcriptomic analysis, we think \u003cem\u003eAh02g107800\u003c/em\u003e may also have a role in abiotic stress responses in peanut. Hap3 of \u003cem\u003eAhDTF1\u003c/em\u003e leads to a new TAAT box and the disappearance of a DNA methylation site, which results in differences in its gene expression, with upregulation of \u003cem\u003eOsDTF1\u003c/em\u003e expression promoting root elongation under drought conditions, thereby enhancing drought resistance.\u003c/p\u003e \u003cp\u003eIn the drought-associated group, AtMYB15 is the \u003cem\u003eArabidopsis\u003c/em\u003e protein closest to AhDTF1, but with low sequence similarity (Additional file 9). AtMYB15 does not have clear tissue-specific expression (Additional file 10), while both \u003cem\u003eAhDTF1\u003c/em\u003e and \u003cem\u003eAh02g107800\u003c/em\u003e are mainly expressed in underground tissues (e.g., developing pods, roots, and gynophores) and have lower expression in above-ground parts like leaves (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). This suggests that AhDTF1 may be a new type of drought-responsive regulator.\u003c/p\u003e \u003cp\u003eRoot growth in soil must overcome physical limits like compaction and resistance. High expression in root tissues may therefore show abiotic stress resistance. For example, the potato \u003cem\u003ePYL16\u003c/em\u003e gene, which is highly expressed in roots, gives drought resistance [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Since genes often work where they are expressed [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e], the better root growth\u0026mdash;shown by higher root-shoot ratio and root hair density\u0026mdash;seen in \u003cem\u003eAhDTF1\u003c/em\u003e-overexpressing plants under mannitol stress fits with its expression pattern. AhDTF1 seems to give two benefits: promoting root growth under stress and boosting cellular antioxidant ability (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). The lighter seed coat color in transgenic materials may reflect more antioxidant activity. As a geocarpic species, peanut has fertilization above ground but fruit development below ground. The high expression of \u003cem\u003eAh02g107800\u003c/em\u003e and \u003cem\u003eAhDTF1\u003c/em\u003e in below-ground tissues suggests important roles in pod development.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePeanut is a major global oil and cash crop. However, its low reproduction rate and complex allotetraploid genome (AABB, 2n\u0026thinsp;=\u0026thinsp;4x\u0026thinsp;=\u0026thinsp;40) make genetic mapping hard with standard linkage analysis. Even large association studies have difficulty measuring complex traits like drought tolerance. This study shows how combining evolutionary-phylogenetic analysis, transcriptomic profiling, and expression checks can quickly find stress-related regulators. With screening of natural variants, this method can help find better alleles for breeding stress-resistant cultivars. Our work provides a method for finding genes in peanut and genetic material for drought tolerance breeding.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study describes a novel peanut R2R3-MYB transcription factor, AhDTF1, which functions as a nuclear transcriptional activator that positively regulates drought resistance. Transgenic tobacco overexpressing AhDTF1 exhibited significantly enhanced drought tolerance, characterized by elevated activities of antioxidant enzymes (POD, SOD, APX, CAT, GST), increased soluble protein content, and reduced malondialdehyde (MDA) accumulation, indicating mitigated oxidative damage. AhDTF1 activates the expression of key stress-responsive genes, including those encoding antioxidant enzymes and other critical factors such as CAX3 and OAT, thereby coordinating antioxidant defense and osmotic adjustment. Furthermore, AhDTF1 regulates seed coat color and root hair development under abiotic stress, with the Hap3 haplotype associated with longer root length, enhancing stress adaptation. In conclusion, AhDTF1 plays a critical role in plant drought adaptation through MYB-dependent transcriptional regulation of antioxidant and osmotic processes. It provides a dual-tolerance mechanism against drought and oxidative stress, making it a promising candidate gene for improving crop resilience in breeding programs.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003ePlant growth and stress treatments\u003c/h2\u003e \u003cp\u003eSeeds of peanut variety 'Changhua 18' were kept in our laboratory. After soaking in water for 4 h, seeds were sown in a 3:1 (v/v) mix of nutrient soil and vermiculite and grown in a greenhouse at 28\u0026deg;C with a 16-h light/8-h dark period. For \u003cem\u003eArabidopsis thaliana\u003c/em\u003e drought treatments, seeds were sown in the same soil mix and grown at 22\u0026deg;C under 16-h light/8-h dark cycles. After four weeks, water was stopped for 14 days then resumed. For tobacco tests, seeds were sown on full MS medium or MS medium with 100, 200, or 300 mM mannitol. Tobacco plants were kept at 25\u0026deg;C under 16-h light/8-dark conditions.The cultivation of all the aforementioned materials was conducted in the greenhouse at Jiangxi Agricultural University, Nanchang City, Jiangxi Province, China.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSequence and phylogenetic analysis\u003c/h3\u003e\n\u003cp\u003eAhR2R3-MYB sequences were re-checked using the latest peanut genome annotation (Tifrunner.gnm2) as in Wang et al. [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Eleven drought-related AtR2R3-MYB sequences were taken from TAIR [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Multiple sequence alignment was done with MAFFT v7.520 [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Phylogenetic analysis used the Neighbor-Joining method with the JTT model and 1000 bootstrap repeats. Gene annotations came from PeanutBase [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eExpression Analysis\u003c/h2\u003e \u003cp\u003ePeanut expression data were downloaded from PeanutBase [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Drought-stress transcriptome data were from previous studies [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Difference expression data are in Additional file 11. AtMYB15 expression data were from TAIR. Affymetrix ATH1 array data were normalized with the GCOS method and a TGT value of 100 [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFor tobacco gene expression tests, 20-day-old plants were split into control and treatment (100 mM mannitol for 24 h) groups. Leaf tissues were collected, frozen in liquid nitrogen, and stored at \u0026minus;\u0026thinsp;80\u0026deg;C. Total RNA was taken out using RNAiso Plus (Takara Bio Inc.). cDNA was made with the PrimeScript\u0026trade; FAST RT reagent Kit with gDNA Eraser (Takara Bio Inc.). qRT-PCR was done on an ABI 7500 system (Applied Biosystems, USA) with TB Green\u0026reg; Premix Ex Taq\u0026trade; II (Tli RNaseH Plus) under these steps: 95\u0026deg;C for 30 s, then 40 cycles of 95\u0026deg;C for 5 s and 60\u0026deg;C for 34 s. Each reaction was done three times, and relative expression was found with the 2\u0026thinsp;\u0026minus;\u0026thinsp;ΔΔCt method. Gene-specific primers are in Additional file 12.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eSubcellular localization\u003c/h2\u003e \u003cp\u003eThe coding sequence of \u003cem\u003eAhDTF1\u003c/em\u003e was amplified and put into the pCAMBIA1300-EGFP vector to make a 35S:EGFP-AhDTF1 fusion construct. The nuclear marker AtHistone H1.2-TagRFP [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e] was used as a co-transfection control. Constructs were put into tobacco epidermal cells through agroinfiltration and kept at 25\u0026deg;C for 36\u0026ndash;48 h. GFP and RFP signals were seen with a confocal laser-scanning microscope (Olympus FV4000, Japan). Primers are in Additional file 13.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eTranscriptional activation assay\u003c/h2\u003e \u003cp\u003eThe full-length CDS of \u003cem\u003eAhDTF1\u003c/em\u003e and its shortened parts (N-terminal: 1\u0026ndash;161 aa; middle: 143\u0026ndash;221 aa; C-terminal: 195\u0026ndash;284 aa) were cloned into the pGBKT7 vector. Each construct was put with pGADT7 into Y2H Gold yeast cells and placed on SD/-Trp-Leu and SD/-Trp-Leu/X-α-gal/AbA media. Yeast transformation and growth followed the Clontech manual. Primers are in Table S5.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eVectorconstructs and plant transformation\u003c/h2\u003e \u003cp\u003eThe full-length CDS of AhDTF1 was cloned into the pQB-V3 vector (with attL1/attL2 sites) and then moved to pGWB511 via Gateway\u0026trade; LR Clonase\u0026trade; II recombination (Invitrogen, Cat. #11791020). Nicotiana tabacum leaf discs were transformed with Agrobacterium tumefaciens strain EHA105. \u003cem\u003eArabidopsis thaliana\u003c/em\u003e (Col-0) was transformed by floral dip with A. tumefaciens strain GV3101. Transgenic plants were selected on hygromycin B and checked by PCR. T2 generation lines were used for tests.\u003c/p\u003e \u003cp\u003eFor transient overexpression in peanut, a bacterial suspension (GV3101 with 35S:AhDTF1) was put into the lower leaf surfaces of variety 'Changhua 06'. After 72 h in the dark, plants were treated with drought using 20% PEG. Plants were grown in water at 25\u0026deg;C for 14 days before treatment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of soluble protein and MDA content\u003c/h2\u003e \u003cp\u003eSoluble protein content was found with the Bradford method [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e] with BSA as standard. For MDA content, leaf samples (0.5 g) were ground in 0.6% thiobarbituric acid and heated in a boiling water bath for 15 min. After centrifugation at 12,000 \u0026times; g for 15 min, absorbance was read at 450 nm, 532 nm, and 600 nm [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eAntioxidant enzyme activity tests\u003c/h2\u003e \u003cp\u003eEnzyme extraction was done as in Sekmen et al. [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e] with changes. Leaf tissues (0.5 g) were ground in 10 mL phosphate buffer (PBS, pH 7.8) and spun at 10,000 rpm for 15 min at 4\u0026deg;C. The supernatant was the enzyme extract. SOD activity was measured by how it stopped nitroblue tetrazolium (NBT) photoreduction [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. The reaction mix had 130 mM PBS (pH 7.8), 130 mM methionine, 750 \u0026micro;M NBT, 100 \u0026micro;M EDTA, 20 \u0026micro;M riboflavin, and 20 \u0026micro;L enzyme extract. After 30 min light (4000 lux), absorbance was read at 560 nm. POD activity was tested with guaiacol as substrate [61]. One unit of POD activity was set as an increase of 0.01 in absorbance per minute at 470 nm.\u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAhDTF1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDrought Tolerance Factor 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePOD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePeroxidase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSOD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSuperoxide dismutase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAPX\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eascorbate peroxidase, CAT:Catalase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGST\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGlutathione S-transferase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMDA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emalondialdehyde\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eWT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ewild-type\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCAX3\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003evacuolar cation/proton exchanger 3\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eand \u003cem\u003eOAT\u003c/em\u003e (ornithine aminotransferase). ABA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eabscisic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eROS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ereactive oxygen\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTFs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etranscription factors\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eABREs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eABA-responsive elements\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHTH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehelix-turn-helix\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor\u0026apos;s Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLYW, HCM, and JHF designed the project. SJW,YWY, and JBH performed data analysis. SJW, YWY, SXW, JBH and YL conducted the experiments. SJW wrote the original manuscript. LYW and HCM contributed to the interpretation of data. LYW and HCM revised the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Prof. Juncheng Zhang from Hangzhou Normal University, for supplying the vectors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Jiangxi Province Key R\u0026amp;D Program Project (20243BBH81028), the the Jiangxi Agriculture Research System (No.JCARS-02), Special Topics on Sustainable Agricultural Science and Technology Innovation of Xinjiang Academy of Agricultural Sciences (xjnkywdzc-2023001-27), and the National Natural Science Foundation of China (32160433 and 32160101).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData is provided within the manuscript or supplementary information files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGupta A, Rico-Medina A, Ca\u0026ntilde;o-Delgado AI. The physiology of plant responses to drought. Science. 2020;368:266\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNakashima K, Tran LS, Van Nguyen D, Fujita M, Maruyama K, et al. Functional analysis of a NAC-type transcription factor OsNAC6 involved in abiotic and biotic stress-responsive gene expression in rice. Plant J. 2007;51:617\u0026ndash;30.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChoudhury FK, Rivero RM, Blumwald E, Mittler R. Reactive oxygen species, abiotic stress and stress combination. Plant J. 2017;90:856\u0026ndash;67.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTardieu F, Simonneau T, Muller B. The physiological basis of drought tolerance in crop plants: a scenario-dependent probabilistic approach. Annu Rev Plant Biol. 2018;69:733\u0026ndash;59.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHa Y, Shang Y, Nam KH. Brassinosteroids modulate ABA-induced stomatal closure in \u003cem\u003eArabidopsis\u003c/em\u003e. J Exp Bot. 2016;67:6297\u0026ndash;308.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrodribb TJ, McAdam SA. Evolution of the stomatal regulation of plant water content. Plant Physiol. 2017;174:639\u0026ndash;49.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBatool S, Uslu VV, Rajab H, Ahmad N, Waadt R, et al. Sulfate is incorporated into cysteine to trigger ABA production and stomatal closure. Plant Cell. 2018;30:2973\u0026ndash;87.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRellan-Alvarez R, Lobet G, Dinneny JR. Environmental control of root system biology. Annu Rev Plant Biol. 2016;67:619\u0026ndash;42.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDinneny JR. Developmental responses to water and salinity in root systems. Annu Rev Cell Dev Biol. 2019;35:239\u0026ndash;57.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRanjan A, Sinha R, Singla-Pareek SL, Pareek A, Singh AK. Shaping the root system architecture in plants for adaptation to drought stress. Physiol Plant. 2022;174:e13651.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFabregas N, Lozano-Elena F, Blasco-Esc\u0026aacute;mez D, Tohge T, Mart\u0026iacute;nez-And\u0026uacute;jar C, et al. Overexpression of the vascular brassinosteroid receptor BRL3 confers drought resistance without penalizing plant growth. Nat Commun. 2018;9:4680.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen T, Li W, Hu X, Guo J, Liu A, et al. A cotton MYB transcription factor, GbMYB5, is positively involved in plant adaptive response to drought stress. Plant Cell Physiol. 2015;56:917\u0026ndash;29.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang N, Zhang W, Qin M, Li S, Qiao M, et al. Drought tolerance conferred in soybean (\u003cem\u003eGlycine max\u003c/em\u003e. L) by GmMYB84, a novel R2R3-MYB transcription factor. Plant Cell Physiol. 2017;58:1764\u0026ndash;76.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBailey-Serres J, Parker JE, Ainsworth EA, Oldroyd GED, Schroeder JI. Genetic strategies for improving crop yields. Nature. 2019;575:109\u0026ndash;18.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCui X, Tang M, Li L, Chang J, Yang X, et al. Expression patterns and molecular mechanisms regulating drought tolerance of soybean [\u003cem\u003eGlycine max\u003c/em\u003e (L.) Merr.] conferred by transcription factor gene \u003cem\u003eGmNAC19\u003c/em\u003e. Int J Mol Sci. 2024;25:1234.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCominelli E, Galbiati M, Tonelli C. Transcription factors controlling stomatal movements and drought tolerance. Transcription. 2010;1:41\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHussain Q, Asim M, Zhang R, Khan R, Farooq S, et al. Transcription factors interact with ABA through gene expression and signaling pathways to mitigate drought and salinity stress. Biomolecules. 2021;11:1159.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShinozaki K, Yamaguchi-Shinozaki K. Molecular responses to dehydration and low temperature: differences and cross-talk between two stress signaling pathways. Curr Opin Plant Biol. 2000;3:217\u0026ndash;23.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYamaguchi-Shinozaki K, Shinozaki K. Organization of cis-acting regulatory elements in osmotic- and cold-stress-responsive promoters. Trends Plant Sci. 2005;10:88\u0026ndash;94.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAhn TS, Ka JO, Lee GH, Song HG. Microcosm study for revegetation of barren land with wild plants by some plant growth-promoting rhizobacteria. J Microbiol Biotechnol. 2007;17:52\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCutler SR, Rodriguez PL, Finkelstein RR, Abrams SR. Abscisic acid: emergence of a core signaling network. Annu Rev Plant Biol. 2010;61:651\u0026ndash;79.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTuteja N. Abscisic acid and abiotic stress signaling. Plant Signal Behav. 2007;2:135\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang W, Liu XD, Chi XJ, Wu CA, Li YZ, et al. Dwarf apple MbDREB1 enhances plant tolerance to low temperature, drought, and salt stress via both ABA-dependent and ABA-independent pathways. Planta. 2011;233:219\u0026ndash;29.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJoshi R, Wani SH, Singh B, Bohra A, Dar ZA, et al. Transcription factors and plants response to drought stress: current understanding and future directions. Front Plant Sci. 2016;7:1029.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHu W, Huang C, Deng X, Zhou S, Chen L, et al. TaASR1, a transcription factor gene in wheat, confers drought stress tolerance in transgenic tobacco. Plant Cell Environ. 2013;36:1449\u0026ndash;64.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKhan SA, Li MZ, Wang SM, Yin HJ. Revisiting the role of plant transcription factors in the battle against abiotic stress. Int J Mol Sci. 2018;19:1634.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOgata K, Kanei-Ishii C, Sasaki M, Hatanaka H, Nagadoi A, et al. The cavity in the hydrophobic core of MYB DNA-binding domain is reserved for DNA recognition and trans-activation. Nat Struct Biol. 1996;3:178\u0026ndash;87.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDubos C, Stracke R, Grotewold E, Weisshaar B, Martin C, et al. MYB transcription factors in \u003cem\u003eArabidopsis\u003c/em\u003e. Trends Plant Sci. 2010;15:573\u0026ndash;81.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu J, Osbourn A, Ma P. MYB transcription factors as regulators of phenylpropanoid metabolism in plants. Mol Plant. 2015;8:689\u0026ndash;708.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi J, Han G, Sun C, Sui N. Research advances of MYB transcription factors in plant stress resistance and breeding. Plant Signal Behav. 2019;14:1613131.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang X, Niu Y, Zheng Y. Multiple functions of MYB transcription factors in abiotic stress responses. Int J Mol Sci. 2021;22:6125.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCominelli E, Galbiati M, Vavasseur A, Conti L, Sala T, et al. A guard-cell-specific MYB transcription factor regulates stomatal movements and plant drought tolerance. Curr Biol. 2005;15:1196\u0026ndash;200.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOh JE, Kwon Y, Kim JH, Noh H, Hong SW, et al. A dual role for MYB60 in stomatal regulation and root growth of \u003cem\u003eArabidopsis thaliana\u003c/em\u003e under drought stress. Plant Mol Biol. 2011;77:91\u0026ndash;103.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCominelli E, Sala T, Calvi D, Gusmaroli G, Tonelli C. Over-expression of the \u003cem\u003eArabidopsis\u003c/em\u003e AtMYB41 gene alters cell expansion and leaf surface permeability. Plant J. 2008;53:53\u0026ndash;64.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSeo PJ, Lee SB, Suh MC, Park MJ, Go YS, et al. The MYB96 transcription factor regulates cuticular wax biosynthesis under drought conditions in \u003cem\u003eArabidopsis\u003c/em\u003e. Plant Cell. 2011;23:1138\u0026ndash;52.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee SB, Kim H, Kim RJ, Suh MC. Overexpression of \u003cem\u003eArabidopsis MYB96\u003c/em\u003e confers drought resistance in \u003cem\u003eCamelina sativa\u003c/em\u003e via cuticular wax accumulation. Plant Cell Rep. 2014;33:1535\u0026ndash;46.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee SB, Suh MC. Cuticular wax biosynthesis is up-regulated by the MYB94 transcription factor in \u003cem\u003eArabidopsis\u003c/em\u003e. Plant Cell Physiol. 2015;56:48\u0026ndash;60.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee SB, Kim HU, Suh MC. MYB94 and MYB96 additively activate cuticular wax biosynthesis in \u003cem\u003eArabidopsis\u003c/em\u003e. Plant Cell Physiol. 2016;57:2300\u0026ndash;11.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNakabayashi R, Yonekura-Sakakibara K, Urano K, Suzuki M, Yamada Y, et al. Enhancement of oxidative and drought tolerance in \u003cem\u003eArabidopsis\u003c/em\u003e by overaccumulation of antioxidant flavonoids. Plant J. 2014;77:367\u0026ndash;79.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang F, Kong W, Wong G, Fu L, Peng R, et al. AtMYB12 regulates flavonoids accumulation and abiotic stress tolerance in transgenic \u003cem\u003eArabidopsis thaliana\u003c/em\u003e. Mol Genet Genomics. 2016;291:1545\u0026ndash;59.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang P, Wang R, Yang X, Ju Q, Li W, et al. The R2R3-MYB transcription factor AtMYB49 modulates salt tolerance in \u003cem\u003eArabidopsi\u003c/em\u003es by modulating the cuticle formation and antioxidant defence. Plant Cell Environ. 2020;43:1925\u0026ndash;43.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu Y, Shen Y, Liang M, Zhang X, Xu J, et al. Identification of peanut AhMYB44 transcription factors and their multiple roles in drought stress responses. Plants (Basel). 2022;11:300.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrasileiro ACM, Morgante CV, Araujo ACG, Leal-Bertioli SCM, Silva AK, et al. Transcriptome profiling of wild Arachis from water-limited environments uncovers drought tolerance candidate genes. Plant Mol Biol Rep. 2015;33:1876\u0026ndash;92.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao N, Cui S, Li X, Liu B, Deng H, et al. Transcriptome and co-expression network analyses reveal differential gene expression and pathways in response to severe drought stress in peanut (\u003cem\u003eArachis hypogaea\u003c/em\u003e L). Front Genet. 2021;12:672884.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRen J, Guo P, Zhang H, Shi X, Ai X, et al. Comparative physiological and coexpression network analyses reveal the potential drought tolerance mechanism of peanut. BMC Plant Biol. 2022;22:460.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu X, Su L, Li L, Zhang Z, Li X, et al. Transcriptome profiling reveals characteristics of hairy root and the role of \u003cem\u003eAhGLK1\u003c/em\u003e in response to drought stress and post-drought recovery in peanut. BMC Genomics. 2023;24:119.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang S, Xu Z, Yang Y, Ren W, Fang J, et al. Genome-wide analysis of R2R3-MYB genes in cultivated peanut (\u003cem\u003eArachis hypogaea\u003c/em\u003e L.): Gene duplications, functional conservation, and diversification. Front Plant Sci. 2023;14:1102174.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu J, Lee YJ, Liu B. Establishment of a mitotic model system by transient expression of the D-type cyclin in differentiated leaf cells of tobacco (\u003cem\u003eNicotiana benthamiana\u003c/em\u003e). New Phytol. 2020;226:1213\u0026ndash;20.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJiang Z, Zhou X, Li R, Michal JJ, Zhang S, et al. Whole transcriptome analysis with sequencing: methods, challenges and potential solutions. Cell Mol Life Sci. 2015;72:3425\u0026ndash;39.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJiang C, Gu X, Peterson T. Identification of conserved gene structures and carboxy-terminal motifs in the MYB gene family of \u003cem\u003eArabidopsis\u003c/em\u003e and \u003cem\u003eOryza sativa\u003c/em\u003e L. ssp. indica. Genome Biol. 2004;5:R46.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYao P, Zhang C, Bi Z, Liu Y, Liu Z, Wei J, Su X, Bai J, Cui J, Sun C. Overexpression of potato \u003cem\u003ePYL16\u003c/em\u003e gene in tobacco enhances the transgenic plant tolerance to drought stress. Int J Mol Sci. 2024;25:8644.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLawson MJ, Church GM. Tissue-specific gene expression: Mechanisms and evolutionary implications. Nat Rev Genet. 2022;22:305\u0026ndash;22.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKatoh K, Rozewicki J, Yamada KD. MAFFT online service: multiple sequence alignment, interactive sequence choice and visualization. Brief Bioinform. 2019;20:1160\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eClevenger J, Chu Y, Scheffler B, Ozias-Akins P. A developmental transcriptome map for allotetraploid \u003cem\u003eArachis hypogaea\u003c/em\u003e. Front Plant Sci. 2016;7:644.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchmid M, Davison TS, Henz SR, Pape UJ, Demar M, et al. A gene expression map of \u003cem\u003eArabidopsis thaliana\u003c/em\u003e development. Nat Genet. 2005;37:501\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248\u0026ndash;54.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHeath RL, Packer L. Reprint of: Photoperoxidation in isolated chloroplasts. I. Kinetics and stoichiometry of fatty acid peroxidation. Arch Biochem Biophys. 2022;726:109248.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSekmen AH, Ozgur R, Uzilday B, Turkan I. Reactive oxygen species scavenging capacities of cotton (\u003cem\u003eGossypium hirsutum\u003c/em\u003e) cultivars under combined drought and heat induced oxidative stress. Environ Exp Bot. 2014;99:141\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGiannopolitis CN, Ries SK. Superoxide dismutases: II. Purification and quantitative relationship with water-soluble protein in seedlings. Plant Physiol. 1977;59:315\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMiao BH, Han XG, Zhang WH. The ameliorative effect of silicon on soybean seedlings grown in potassium-deficient medium. Ann Bot. 2010;105:967\u0026ndash;73.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"peanut, R2R3-MYB, AhDTF1, transcriptional activator, drought tolerance, antioxidant","lastPublishedDoi":"10.21203/rs.3.rs-7702056/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7702056/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003ePeanut (\u003cem\u003eArachis hypogaea\u003c/em\u003e L.), a major oilseed crop globally, suffers significant yield reduction due to drought stress. Here, we identified a novel R2R3-MYB transcription factor AhDTF1 through phylogenetic analysis, comprehensive transcriptomic analysis of drought responses, and tissue expression pattern.\u003c/p\u003e\u003ch2\u003eResult\u003c/h2\u003e \u003cp\u003eAhDTF1 functioned as a nuclear transcriptional activator and positively regulates drought resistance. Transgenic tobacco plants over-expressing \u003cem\u003eAhDTF1\u003c/em\u003e exhibited significantly enhanced drought tolerance, manifested by elevated activities of antioxidant enzymes such as POD, SOD, APX, CAT, and GST, increased soluble protein content, and reduced MDA accumulation compared to WT. Furthermore, AhDTF1 activated the expression of key stress-responsive genes, including \u003cem\u003eAPX\u003c/em\u003e, \u003cem\u003eCAT\u003c/em\u003e, \u003cem\u003ePOD\u003c/em\u003e, \u003cem\u003eSOD\u003c/em\u003e, \u003cem\u003eGST\u003c/em\u003e, \u003cem\u003eCAX3\u003c/em\u003e, and \u003cem\u003eOAT\u003c/em\u003e. AhDTF1 also regulated seed coat color and root hair development under abiotic stress conditions, Hap3 conferring longer root length. These findings demonstrated that AhDTF1 played a critical role in plant drought stress adaptation by coordinating antioxidant defense mechanisms and osmotic adjustment. Our work highlighted AhDTF1 as a promising candidate for crop improvement, providing a dual-tolerance strategy against drought and associated oxidative stresses through MYB-dependent transcriptional regulation.\u003c/p\u003e","manuscriptTitle":"AhDTF1, a novel R2R3-MYB transcription factor, involves in drought tolerance and seed color","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-29 03:40:46","doi":"10.21203/rs.3.rs-7702056/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-15T11:11:24+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-06T12:25:19+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-03T11:38:34+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"40030783532070597215220415595103864180","date":"2026-04-27T10:16:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"245979851266495809453855211817726234004","date":"2026-04-24T01:47:01+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-24T19:15:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"87931886210608153920482984096010852150","date":"2026-01-27T19:21:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"27156635035302884192000378969147106735","date":"2025-12-27T14:47:52+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-25T13:55:02+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-12-18T07:22:41+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-14T15:34:09+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-14T04:05:08+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2025-10-14T04:01:41+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"df538dbd-8d39-48ef-85f5-ca16eea2e2d6","owner":[],"postedDate":"December 29th, 2025","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-15T11:11:24+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-05-15T11:24:26+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-29 03:40:46","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7702056","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7702056","identity":"rs-7702056","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}