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. 2025 Feb 3;26:101. doi: 10.1186/s12864-025-11241-5

Comprehensive genome-wide analysis of ARF transcription factors in orchardgrass (Dactylis glomerata): the positive regulatory role of DgARF7 in drought resistance

Miaoli Wang 1,#, Wen Liu 1,#, Guangyan Feng 1, Gang Nie 1, Zhongfu Yang 1, Feixiang Hao 1, Linkai Huang 1,✉, Xinquan Zhang 1,✉
PMCID: PMC11792575  PMID: 39901077

Abstract

Auxin response factor (ARF), a transcription factor, is crucial in controlling growth, development, and response to environmental stress. Orchardgrass (Dactylis glomerata) is an economically significant, widely cultivated forage grass. However, information on the genome-wide information and functional characterization of ARFs in orchardgrass is limited. This study identified 27 ARF genes based on the orchardgrass genome database. These DgARFs were unevenly distributed across the seven orchardgrass chromosomes and clustered into four classes. Phylogenetic analysis with multispecies of ARF proteins indicated that the ARFs exhibit a relatively conserved evolutionary path. Focusing on hormone signaling responses, DgARF7 demonstrated a potential positive regulatory role in response to 3-indole acetic acid, methyl jasmonate, gibberellin, salicylic acid, and abscisic acid signals. Additionally, exposure to drought stress induced noticeable oscillatory changes in DgARF7 gene. Notably, DgARF7 enhanced drought tolerance through heterologous expression in yeast and overexpression in Arabidopsis. Overexpressed Arabidopsis lines of DgARF7 exhibited a markedly higher relative water content and superoxide dismutase activity, while the malondialdehyde content was significantly decreased compared to wild type under drought stress. DgARF7 also accelerated flowering time by inducing the flowering-related gene expression levels in Arabidopsis. This research provides important insights into the role of DgARF7 in orchardgrass and provides further understanding in molecular breeding.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12864-025-11241-5.

Keywords: ARF gene family, Dactylis glomerata, Expression patterns, Drought stress, Flowering time

Introduction

Auxin is a ubiquitous hormone that plays a crucial role in various stages from embryogenesis to senescence, and is largely utilized in plant stress responses [1, 2]. The Aux/IAA, SAUR, GH3, and ARF gene families, which respond to auxin, are crucial in transmitting auxin signals [3]. Moreover, the ARF gene family, as important transcription factors of plants, is crucial in signal transduction, cellular morphogenesis, and environmental resistance [4, 5]. ARF proteins can bind to AUX/IAA proteins, inhibiting auxin-responsive genes in the absence of auxin. The ARF gene family consists of three domains: the essential N-terminal B3 DNA-binding domain (DBD), a middle region (MR), and a C-terminal dimerization domain (CTD). The DBD is capable of recognizing the AuxRE found in the promoter of the target gene, while MR serves as either an activation domain (AD) or a repression domain (RD). Moreover, CTD facilitates the interaction between ARF and AUX/IAA proteins [6, 7].

The significant functions of ARFs in various growth and physiological processes have prompted their identification and analysis in several organisms, including Arabidopsis thaliana [8], Oryza sativa [9], Phalaenopsis Aphrodite [10], Medicago sativa [11], Gossypium hirsutum [12], Magnolia sieboldii [13], and Solanum lycopersicum [14]. The functional studies of ARFs focus on model plants A. thaliana, O. sativa, and S. lycopersicum. In A. thaliana, double mutants of arf1 and arf2 exhibited delayed flowering onset, senescence of rosette leaves, abscission of floral organs, and ripening of siliques [15]. Double mutants of arf7 and arf19 displayed reduced lateral root growth and irregular gravitropism at the hypocotyl and root development stages [8]. Overexpressing ARF2 increased sensitivity to low-K+ stress [16]. In O. sativa, inhibiting ARF1 expression caused extremely low growth and sterility [17]. Mutants of arf11 had fewer filled seeds, fewer branches, and fewer grains in the panicle [18]. The ARF12 gene regulates phosphate homeostasis [19]. In S. lycopersicum, decreased mRNA expression of SIARF7 resulted in seedless fruits [20]. mSlARF10 transcript accumulation dramatically reduced laminar outgrowth, rendering the leaves almost bladeless [21]. Knockdowns of ARF2 have improved resistance to salt and drought stresses, and SlARF4 knockouts showed improved water deficit resistance [22, 23]. Nevertheless, there have been no documented reports of the ARF gene family in orchardgrass.

Orchardgrass is a widely grown, significant, economically valuable forage grass that contributes greatly to livestock husbandry and environmental restoration [24].Late-flowering varieties in orchardgrass exhibit reduced net herbage accumulation during rotational grazing [25] or decreased survival rates [26]. The increasingly harsh climate has a serious negative impact on the quality and yield of orchardgrass. Thus, breeding novel materials with early flowering time and higher resistance to abiotic stress is of high importance. Numerous studies have demonstrated the importance of ARF transcription factors in controlling plant growth, development, and stress response, making ARFs a crucial gene family for comprehending plant biology [27]. Moreover, the ARF gene family can provide important resources for improving forage grass yield and quality [11]. Thus, this study identified 27 ARF genes in orchardgrass and their physiochemical characteristics, cladograms, gene structures, motifs, and chromosome locations. Furthermore, they also examined the cis-acting elements and expression levels of DgARF genes in response to hormones and abiotic stresses, aiming to elucidate the functions of the ARF gene family in orchardgrass. Last, the study examined if the homologous expression of DgARF7 can enhance drought tolerance in yeast and Arabidopsis, and if its overexpression in Arabidopsis induced the early flowering phenotype. This study provides a better understanding of the ARF gene family in regulating plant growth and development and response to abiotic stresses in orchardgrass.

Materials and methods

Confirmation and protein characterizations of the DgARFs

The genome data and total protein sequences of orchardgrass were obtained from NCBI (https://www.ncbi.nlm.nih.gov/) using the accession number PRJNA471014. The ARF protein sequences for Arabidopsis thaliana, Oryza sativa, Triticum aestivum, Zea mays, Raphanus sativus, Solanum tuberosum, Solanum lycopersicum, Vigna radiata, Medicago truncatula, Malus domestica, and Prunus mume were sourced from the Plant transcription factor database (https://planttfdb.gao-lab.org/), and their protein sequences were shown in Table S1. The ARF protein sequences of Oryza sativa were utilized as queries for potential DgARF proteins using the BLASTP tool in NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Furthermore, hidden Markov model (HMM) profiles containing the B3 DNA binding (Pfam 02362), Auxin Resp (Pfam 06507), and PB1 domains (Pfam 02309) were used to confirm the potential DgARF proteins.

ProtParam tool was applied to investigate the protein characteristics of the DgARFs, encompassing aspects such as amino acids length, molecular weight, theoretical pI, instability index, aliphatic index, and grand average of hydropathicity (https://web.expasy.org/protparam/). The secondary structure of ARF proteins in orchardgrass were explored using SOPMA (https://nPsa-prabi.ibcp.fr/cgi-bin/nPsa_automat.pl?page=nPsa_sopma.html). Finally, the putative DgARF protein domains were obtained using the Conserved Domain Search tool (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi.).

Phylogenetics, gene structures, motifs and chromosomal locations analysis

The phylogenetic tree was created through maximum likelihood (ML) analysis using the MEGA 7.0 software, incorporating 1,000 bootstrap iterations [28]. The phylogenetic trees were labeled and colored via the iTOL (https://itol.embl.de/itol.cgi). The intron–exon structure and chromosome location were analyzed using TBtools, and motifs analysis was performed using the MEME Suite (http://meme-suite.org/tools/meme).

Analysis of cis-acting elements on DgARFs promoter

The authors chose a 2000 bp sequence located upstream of the DgARFs to examine the cis-regulatory elements acquired from the PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/).

Expression patterns of ARFs in various hormone treatments and abiotic stresses

The “2006–1” seeds were cultured in pots containing quartz and were watered with Hoagland. The hormone of 100 μM IAA (3-indole acetic acid), 100 μM ABA (abscisic acid), 100 μM MeJA (methyl jasmonate), 100 μM SA (salicylic acid), and 100 μM GA (gibberellin) were sprayed on the leaves of 28 d seedlings, respectively. For abiotic stress, the seedlings were exposed to 20% Polyethylene glycol (PEG) 6000 (W/V) and 4℃ low temperatures. Next, leaf samples were gathered at 0, 1, 2, and 4 h after treatment. Each time point and treatment had three biological replicates. The samples were immediately frozen in liquid nitrogen and kept at -80 °C for quantitative real-time PCR (qRT-PCR) examination.

Subcellular localization of DgARF7

DgARF7 was inserted into the pAN580-35S-EGFP vector to estimate its location, and the specific primers were listed in Table S2. Black pAN580-35S-EGFP served as the control vector. The fusion and control vector were transfected into rice protoplasts following a previous procedure [29]. The whereabouts of DgARF7 were visualized using the Nikon C2-ER laser confocal microscope system, with a 488 nm excitation and a 510 nm emission.

Yeast transformation and validation of drought stress

The DgARF7 was integrated into the pYES2 vector using the related primers (Table S2). Next, the fusion and empty vectors were transferred to the yeast strain INVScI from Weidi Biotechnology Co., Ltd, Shanghai, China. We selected 2.0 and 2.5 mol/L sorbitol as the experimental group to simulate drought conditions, while 0 mol/L sorbitol as control. The procedure of experiment was described as in the previous research [30].

The transformation of DgARF7 in Arabidopsis

The DgARF7 was turned into the pCAMBIA1300-35S-GUS vector (Table S2). The modified plasmid was incorporated into GV3101 and transferred into Arabidopsis using the floral dip method [31]. Transgenic Arabidopsis plants were chosen using 1/2 Murashige and Skoog (MS) medium with 25 μg/mL hygromycin and confirmed through qRT-PCR analysis.

Drought treatment of Arabidopsis

Mannitol or PEG treatment were conducted to imitate drought circumstances to explore the function of DgARF7 in the drought resistance. Wild-type (WT) and DgARF7 transgenic seeds were surfaced-sterilized and sown on 1/2 MS medium for 5 days and then transferred to 1/2 MS medium containing 250 mM mannitol for drought stress [32]. WT and DgARF7 transgenic seedlings in 1/2 MS medium served as a control. The root length of two weeks-old seedlings were measured and photographed. Twenty-one-day-old WT and DgARF7-overexpressing Arabidopsis plants were treated with 20% PEG 6000 solution and incubated for 5 days [33]. In this time, levels of malondialdehyde (MDA), superoxide dismutase (SOD), peroxidase (POD) and total Chlorophyll were assessed using specific assay kits from Grace Biotechnology Co., Ltd., Suzhou, China. The relative water content and relative electrolyte leak were determined using a previously described method [34]. The experiment was performed three times.

qRT-PCR analysis

We used an RNA kit (Magen, Guangzhou, China) and MonScript™ RTIII All-in-One Mix with dsDNase (Monad, Suzhou, China) to extract total RNA and synthesize cDNA, respectively. Next, gene expression was measured with qRT-PCR using the Taq SYBR® Green qPCR Premix (Yugong, Lianyungang, China). The 2−∆∆Ct method [35] was utilized to calculate the expression levels of selected genes. The orchardgrass reference genes were β-actin and GADPH, while those for Arabidopsis were Actin and Actin2 (Table S3).

Results

Identification of ARF gene family in orchardgrass

This research identified 27 ARF genes, unevenly distributed across the seven chromosomes of the orchardgrass genome (Fig. S1; Table S4). The DgARF proteins varied significantly in size (254—1164 amino acids) and molecular weight (28.59—129.44 KDa) (Table S5). Notably, their theoretical isoelectric points varied from 4.82 to 9.35. The instability index of all DgARF proteins exceeds 40, except for DgARF13b, and their grand average hydrophilicity is negative, indicating a tendency towards hydrophilicity and instability in aqueous environments. The secondary structure of ARF proteins included alpha helix, extended strand, beta turn, and random coil (Table S5). Among these components, the random coil constituted the largest proportion (approximately 43.04—66.1%), followed by alpha helix (14.86—35.75%), extended strand (14.29—19.48%), and beta turn (3.68—7.88%). Moreover, DgARF2-3, 8, 10a, 10b, 13b, 14–15, 18, 22, and DgARF24b were absent in the CTD domain, and DgARF13a was absent in the ARF domain.

Analysis of phylogenetic trees, gene structures, and motifs of ARF gene family in orchardgrass

The 27 ARF proteins were categorized into four distinct classes (I, IIa, IIb, and III), aligning with the classification in the rice ARF gene family [9] (Fig. 1). The respective gene structures and motifs further supported this categorization. The gene structures revealed different exon numbers (2 – 14) in the various classes. Class I proteins had 14 exons, except DgARF24b, which had five. Class IIa had 13 and 14 exons, whereas class IIb had 9 to 11. Class III exhibited the lowest exon numbers (2 – 3). Furthermore, motif distribution showed that all DgARFs had motif 2, 4, 5, and 7. Motif 6 was absent in the class IIb DgARF proteins, and motif 10 was absent in the class III DgARF proteins. Motif 10 occurred twice in some class I and IIa ARF proteins, including DgARF1, DgARF5, DgARF9, DgARF12, and DgARF24a. Similar exon numbers and unique motif features within the same class or subclass prove an evolutionary connection among ARF genes in orchardgrass.

Fig. 1.

Fig. 1

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Phylogenetic tree, gene structure, and motifs of the ARF gene family in orchardgrass

Phylogenetic analysis of the ARF gene family

The phylogenetic analysis incorporated ARF protein sequences from 12 different species were divided into four classes (Fig. 2). The four classes are also divided into nine subclasses. Class IIa had more ARFs than other classes. For the evolutionary analysis of ARFs in Gramaceae, DgARFs always congregated with Triticum aestivum and Oryza sativa rather than Zea mays, indicating that it had closer genetic relationship with Triticum aestivum and Oryza sativa. The subclasses of Class I-1 and Class IIb-1 did not include ARFs of Dactylis glomerata, Triticum aestivum, Oryza sativa, and Zea mays. Each class contained ARFs of Gramaceae, Cruciferous, Leguminosae, Solanaceae and Rosaceae, reflecting the conservation of the ARF gene family in different species.

Fig. 2.

Fig. 2

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The phylogenetic tree of the ARF gene family among different plant species. The red font represents the ARF protein of orchardgrass. The ARF proteins include five families: Gramaceae (Dactylis glomerata, Triticum aestivum, Oryza sativa, and Zea mays), Cruciferous (Arabidopsis thaliana and Raphanus sativus), Leguminosae (Medicago truncatula and Vigna radiata), Solanaceae (Solanum tuberosum and Solanum lycopersicum) and Rosaceae (Malus domestica and Prunus mume)

Expression pattern analysis of DgARFs under different hormone treatments and abiotic stresses

The cis-acting regulatory elements in the promoter regions of DgARF genes are primarily involved in responses to light, hormones (auxin, gibberellin, MeJA, salicylic acid, and abscisic acid), stress (drought, low temperature, and anaerobic), and tissue specificity (meristem, root, endosperm, and seed) (Fig. S2a). Light response elements were the most prevalent, followed by MeJA and abscisic acid elements (Fig. S2b). However, gibberellin response elements were absent in the class IIb DgARFs, while salicylic acid response elements were missing in the class IIb and III DgARFs. Anaerobic induction and drought-inducibility elements constituted the largest number of stress response regulatory elements. These findings suggest that DgARFs significantly adapt orchardgrass to light, hormonal signals, and stress conditions.

Ten selected DgARF genes were analyzed using qRT-PCR after subjection to different treatments: IAA, MeJA, GA, SA, ABA, PEG, and cold temperature to elucidate their involvement of DgARFs in hormone signaling and abiotic stress responses (Fig. 3). Under IAA treatment, DgARF4, 7, 9, 12, 15, 21, 22, and 24a were consistently upregulated at 1, 2, and 4 h, contrasting with DgARF5, which was significantly downregulated. Under MeJA treatment, DgARF7 and DgARF12 were upregulated at all time points. Under GA treatment, DgARF5, DgARF7, and DgARF12 were upregulated at various intervals, while DgARF9 and DgARF17 remained unchanged. Under ABA treatment, DgARF4 was upregulated at 1 h and 4 h, while DgARF7, DgARF21, DgARF22, DgARF24a were upregulated at 4 h. Under SA treatment, the gene expression levels of DgARF7, DgARF12, DgARF15 and DgARF21 were higher at 1, 2, and 4 h than 0 h.

Fig. 3.

Fig. 3

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qRT-PCR expression of DgARF genes under different hormone treatments and abiotic stresses. Bars represent the mean value ± standard deviation (SD) (n = 3). One-way ANOVA was used to determine significant differences in the data. The different letters above the bars indicate significant differences among the time points (P < 0.05)

Contrary to their response to hormone treatments, most of the selected ARF family genes exhibited pronounced oscillatory changes under PEG stress. For example, DgARF7 significantly decreased at 1 h and 2 h after exposure to PEG stress, followed by a marked increase at 4 h. For cold treatment, DgARF4, DgARF12, and DgARF17 were upregulated at 2 h and 4 h. DgARF21 were downregulated at 1, 2, and 4 h. These results showed that DgARF7 plays an important role in response to IAA, MeJA, GA, SA, ABA and PEG treatments.

DgARF7 can enhance drought tolerance in yeast strains

After noticing pronounced vibration in DgARF7 with 20% PEG treatment, we aimed to clarify the functional roles of DgARF7 in drought stress response. The fluorescence signal showed that DgARF7 protein was localized in the nucleus (Fig. 4). Furthermore, we transferred the DgARF7 gene into pYES2 vector to analyze its drought resistance (Fig. 5). Under optimal growth conditions, no significant differences were observed between the growth rates of the transformed and the control yeast strains. The pYES2-DgARF7 transformed yeast showed improved resilience, with higher cell recovery rates at 10,000- and 100,000-fold dilutions than the control in 2.0 M sorbitol. At 2.5 M sorbitol, the transformed yeast showed higher cell recovery in 10-, 100-, and 1000-fold dilutions than the control yeast. Consequently, this study hypothesized that the heterologous expression of DgARF7 enhances drought tolerance in yeast transformants.

Fig. 4.

Fig. 4

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The subcellular localization of DgARF7 protein in rice protoplasts. Images from left to right indicate the green florescent protein, bright, and merge fields. Scale bar = 10 μm

Fig. 5.

Fig. 5

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Heterologous expression of DgARF7 enhances drought tolerance in transformant yeast (INVSc1). INVScI yeast cells were dotted on YPG medium in 2 μL aliquots and were diluted ten times using ddH2O

The phenotypes of overexpressing DgARF7 in Arabidopsis

This study overexpressed DgARF7 in wild-type (WT) Arabidopsis using the 35S promoter through Agrobacterium-mediated transformation to investigate its function. Transgenic plants overexpressing DgARF7 exhibited flowering earlier than WT Arabidopsis, prompting the investigation of key genes involved in flowering time regulation (AtFLC, AtLFY, AtAP1, and AtSOC1) in the WT and DgARF7 -transgenic plants (Fig. 6c). The findings revealed a notable reduction in the expression of the negative flowering time regulating gene, AtFLC, in the DgARF7-transgenic than the WT. In contrast, the promoted flowering time genes AtLFY, AtAP1, and AtSOC1 were significantly upregulated in DgARF7-transgenic plants compared to WT. These findings indicate that DgARF7 can regulate these genes, thus accelerating flowering in transgenic Arabidopsis.

Fig. 6.

Fig. 6

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Overexpressing DgARF7 can accelerate flowering time in transgenic Arabidopsis. a The phenotypes of overexpressing DgARF7 T3 homozygous lines and WT plants. b The flowering time and the number of rosettes leaves of the three overexpression DgARF7 lines and WT plants. c The relative expression of the flowering time-related genes at 21 d. Error bar represents the SD (n = 3) in qRT-PCR analysis and SD (n = 10) in the flowering time and number of rosette leaves. One-way ANOVA was used to determine significant differences in the data. The letters above the bars indicate significant differences among the time points (P < 0.05)

DgARF7 can enhance drought tolerance in Arabidopsis

Further investigations into Arabidopsis seedlings revealed that lines OE6 and OE8 exhibited longer root lengths than the WT plants under 250 mM mannitol treatment, indicating overexpressed DgARF7 can enhanced drought resilience (Fig. 7a and b). Physiological parameters of WT and overexpressed DgARF7 plants were evaluated under normal and 20% PEG treatment (Fig. 8). Overexpressed lines exhibited a markedly higher relative water content than WT plants when subjected to PEG treatment (Fig. 8b). Moreover, the levels of MDA in OE2, OE6, and OE8 were notably lower than those of WT plants, both under normal and PEG situations (Fig. 8d). Although the SOD levels in OE2, OE6, and OE8 were lower than in the WT plants under normal conditions, they had a higher level in overexpressed DgARF7 plants than in WT plants under PEG treatment (Fig. 8e). Additionally, the POD and chlorophyll levels in the overexpressed DgARF7 plants were slightly higher than those in WT plants under normal and PEG treatment (Fig. 8f and g).

Fig. 7.

Fig. 7

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DgARF7 enhances drought tolerance in overexpressed Arabidopsis seedlings. a The phenotypes of WT and DgARF7 transgenic lines subjected to 1/2 MS (Control) and 250 mM Mannitol. b The root lengths of WT and DgARF7 transgenic lines subjected to control and 250 mM Mannitol. The bars represent the mean value ± standard deviation (SD) (n = 10). One-way ANOVA was used to determine significant differences in the data. The different letters above the bars indicate significant differences between WT and DgARF7 transgenic plants (P < 0.05)

Fig. 8.

Fig. 8

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DgARF7 transgenic Arabidopsis plants positively regulate drought stress. a The phenotypes of WT and overexpressed DgARF7 plants in normal and 20% PEG treatment. b-g The physiological parameters include relative electrolyte leakage, relative water content, MDA content, SOD, POD and total chlorophyll content under control and PEG conditions. The bars represent the mean value ± standard deviation (SD) (n = 3). One-way ANOVA was used to determine significant differences in the data. The different letters above the bars indicate significant differences between WT and DgARF7 transgenic plants (P < 0.05)

Discussion

This investigation identified 27 ARF genes in orchardgrass. The number of ARFs varies greatly among species and does not necessarily correlate with genome size. For instance, Phalaenopsis Aphrodite has 16 ARF genes [10], Arabidopsis thaliana has 23 [8], Oryza sativa has 25 [9], Glycine max has 51 [36], and Medicago sativa has 81 [11]. The 27 ARF proteins from this study were categorized into four classes (I, IIa, IIb, III) and contained three conserved domains (DBD, ARF, and CTD). However, the CTD domain was absent in classes IIb and III (except for DgARF13a), indicating that these ARFs have lost the capability to mediate the interaction of the ARF domain with AUX/IAA proteins [37]. The absence of CTD may cause ARFs to interact with other transcription factors instead of AUX/IAA, resulting in auxin insensitivity and functioning in an auxin-independent manner as with AtARF3 in Arabidopsis [38, 39].

Gene structure and motifs are crucial factors in gene family evolution. In this study, the classes had 2 to 14 exons, each containing a comparable number. Class I was particularly stable (at 14 exons) except DgARF24b, suggesting a functional redundancy within this class. Each class also displayed unique motif features. For example, motif 6 was absent in class IIb, and motif 10 was absent in class III, suggesting distinct evolutionary paths and functions among the different DgARF classes. The classification of 12 different species ARF genes into four classes. For the evolutionary analysis of ARFs in Gramaceae, DgARFs had closer genetic relationships with Triticum aestivum and Oryza sativa rather than Zea mays. Each classification contained ARF proteins of 12 different species underscores ARF gene family relative conservatism, consistent with findings on flax ARFs [40].

The regulatory function of genes is considered to be positively correlated with the cis-acting elements [41]. In orchardgrass, the promoter region of the ARF genes were mainly linked to light, hormone, and abiotic stress responses, consistent with ARF gene family of other species [42]. This research investigated how abiotic stresses, and external hormones affect the expression of DgARFs, aiming to understand the role of DgARFs in responses to abiotic stresses and phytohormones. Auxin concentration controls ARF activity, and MeJA is the predominant cis-acting element for hormone response in orchardgrass. Therefore, this study assessed the impact of exogenous IAA and MeJA hormones on DgARF gene expression. Eight out of the ten selected DgARF genes had significantly high expression levels under IAA treatment, consistent with a previous finding where IAA treatment upregulated most ARF genes in Brachypodium distachyon [43], Linum usitatissimum [40]. MeJA, modulates the biosynthesis of other phytohormones and boosts natural disease resistance in plants against pathogen and abiotic stresses [44, 45]. Under MeJA treatment, DgARF7 and DgARF12 had higher expression compared to 0 h, agreeing with previous reports for ARF in Salvia miltiorrhiza [46].

Drought is a prevalent issue that adversely affects the economy. Approximately 40% of the total global land area is experiencing drought stress [47]. ARFs control soluble sugar content, facilitate root development, and uphold chlorophyll levels during drought stress, aiding plants in adapting to this stress. In this study, most of the selected ARF genes had obvious oscillatory expression levels under PEG treatment. Furthermore, DgARF7 not only was responsive to various hormone signals including IAA, MeJA, GA, SA, ABA, but also had a marked increase at 4 h under PEG treatment. Past phylogenetic connections of ARF genes in O. sativa and Arabidopsis have offered valuable perspectives for deducing the possible biological roles of DgARF genes [8, 18, 48]. AtARF1, OsARF7, and DgARF7 were clustered together. Mutations in arf1/arf2 are known to negatively regulate senescence, flowering time, stamen development, floral organ abscission, and fruit dehiscence in Arabidopsis [15]. But the function of AtARF1 or OsARF7 in responding drought has not been studied. Hence, it is valuable to explore the role of DgARF7 in responding to drought stress.

DgARF7 is an ARF transcription factor, which is located in the nucleus. Higher cell recovery in yeast under sorbitol treatment suggested that DgARF7 may function as a positive regulator when subjected to drought stress. Thus, this study overexpressed DgARF7 gene in Arabidopsis to further explore the function in responding drought stress. DgARF7 transgenic Arabidopsis plants exhibited increased water content than the WT plants under 20% PEG treatment, suggesting an improved ability to retain moisture, a key role in enhancing drought sufferance. Drought stress can induce lipid peroxidation, damage to membranes, oxidation, and potentially cell death by rapidly increasing reactive oxygen species [49, 50]. Moreover, numerous studies have shown the key role of antioxidant enzymes including SOD and POD in counterbalancing reactive oxygen species in plants [51]. Consistent with these studies, the MDA content was decreased, while SOD and POD levels were increased in DgARF7-overexpressing plants under drought conditions. These results showed that DgARF7-overexpressing plants had higher drought resistance than WT. In addition, we also found other functions in DgARF7-overexpressing Arabidopsis plants. DgARF7 positively regulated flowering time in transgenic Arabidopsis by modulating the expression of AtFLC, AtLFY, AtAP1, and AtSOC1 genes. The transition from vegetative to reproductive development is controlled by multiple environmental and endogenous signals that ultimately converge on key regulators of floral identity: FLC/AP1/SOC1 and LFY [52–54]. ARF transcription factor could regulate multiple independent biological processes because they can interact with distinct sets of substrates. For instance, ARF6 and ARF8 repressed the class 1 KNOX genes to induce the aberrant vascular patterning in petals and delayed the opening of flower buds in Arabidopsis [55]. CRY1 and phyB interact physically with ARF6 and ARF8 through their N-terminal domains in a blue and red light-dependent manner to regulate hypocotyl elongation in Arabidopsis, respectively [56]. ZmARF1 directly targeted ZmLBD1 and positively regulated its transcription to promote root development in maize [57]. ZmARF1 interacts with ZmIAA9 and repress the expression levels of ROS scavenging genes to regulate salt tolerance in maize [58]. Our study showed that DgARF7 could play a positive role in two seemingly independent biological processes: flowering time and drought tolerance. However, the molecular mechanism of DgARF7 in regulating flowering time and drought resistance needs to be explored in future studies.

Conclusion

In this study, we identified 27 ARF genes based on the orchardgrass genome database. These DgARFs clustered into four classes, and the multispecies phylogenetic analysis of ARF proteins indicated that the ARFs exhibit a relatively conserved evolutionary path. DgARF7 plays a positive role in IAA, MeJA, GA, SA and ABA signals. Additionally, it also showed significantly increased at 4 h under drought stress. Heterologous expression DgARF7 enhanced drought tolerance in yeast.

Overexpressed Arabidopsis lines of DgARF7 exhibited a markedly higher relative water content and superoxide dismutase level, while the malondialdehyde content was significantly decreased compared to WT under drought stress. These results showed that DgARF7 may play a positive role in drought tolerance in orchardgrass. This study provides a better understanding in the functions of the ARF gene family in response to hormone signals and drought stresses in orchardgrass.

Supplementary Information

12864_2025_11241_MOESM1_ESM.xlsx (113.7KB, xlsx)

Supplementary Material 1: Supplementary Table S1 The protein sequences of ARF gene family in Arabidopsis thaliana, Oryza sativa, Zea mays, Triticum aestivum, Raphanus sativus, Solanum lycopersicum, Solanum tuberosum, Malus domestica, Prunus mume, Medicago truncatula, and Vigna radiate.

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Supplementary Material 2: Supplementary Table S2 The information of primers used for gene cloning and vector construction.

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Supplementary Material 3: Supplementary Table S3 The primer sequences for qRT-PCR in orchardgrass and Arabidopsis.

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Supplementary Material 4: Supplementary Table S4 The protein sequences of ARF gene family in orchardgrass.

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Supplementary Material 5: Supplementary Table S5 Basic information of ARF gene family in orchardgrass.

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Supplementary Material 6: Supplementary Figure S1 The chromosome location of the ARF gene family in orchardgrass.

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Supplementary Material 7: Supplementary Figure S2 The cis-acting regulatory elements of DgARF gene promoters (2000 bp) in orchardgrass. (a) The distribution of cis-acting regulatory elements of DgARFs. (b) The number of cis-acting regulatory elements of DgARFs.

Acknowledgements

Not applicable.

Authors’ contributions

MLW: writing-original draft preparation, formal analysis and visualization; WL: formal analysis and software; GYF: writing-review and editing and methodology; GN, ZFY and FXH: writing-review and editing; LKH: funding acquisition; XQZ: project administration and funding acquisition. All authors have read and agreed to the published version of the manuscript.

Funding

This research work was supported by China Agriculture Research System of MOF and MARA (CARS-34), National Key Research and Development Program of China (2023YFF1001400), National Natural Science Foundation of China (NSFC31872997), and National Natural Science foundation of China (No. 32071867).

Data availability

The genome data and total protein sequences of orchardgrass were obtained from NCBI BioProject database (https//www.ncbi.nlm.nih.gov/bioproject) using the accession number PRJNA471014. The ARF protein sequences for Arabidopsis thaliana, Oryza sativa, Triticum aestivum, Zea mays, Raphanus sativus, Solanum tuberosum, Solanum lycopersicum, Vigna radiata, Medicago truncatula, Malus domestica, and Prunus mume were sourced from the Plant transcription factor database (https://planttfdb.gao-lab.org/). All data generated or analyzed during this study are included in this article and can be found in the Additional file.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

The authors say they have no conflicting agendas.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Miaoli Wang and Wen Liu contributed equally to this work.

Contributor Information

Linkai Huang, Email: huanglinkai@sicau.edu.cn.

Xinquan Zhang, Email: zhangxq@sicau.edu.cn.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

12864_2025_11241_MOESM1_ESM.xlsx (113.7KB, xlsx)

Supplementary Material 1: Supplementary Table S1 The protein sequences of ARF gene family in Arabidopsis thaliana, Oryza sativa, Zea mays, Triticum aestivum, Raphanus sativus, Solanum lycopersicum, Solanum tuberosum, Malus domestica, Prunus mume, Medicago truncatula, and Vigna radiate.

12864_2025_11241_MOESM2_ESM.xlsx (9.5KB, xlsx)

Supplementary Material 2: Supplementary Table S2 The information of primers used for gene cloning and vector construction.

12864_2025_11241_MOESM3_ESM.xlsx (11.2KB, xlsx)

Supplementary Material 3: Supplementary Table S3 The primer sequences for qRT-PCR in orchardgrass and Arabidopsis.

12864_2025_11241_MOESM4_ESM.xlsx (20.3KB, xlsx)

Supplementary Material 4: Supplementary Table S4 The protein sequences of ARF gene family in orchardgrass.

12864_2025_11241_MOESM5_ESM.xlsx (12.7KB, xlsx)

Supplementary Material 5: Supplementary Table S5 Basic information of ARF gene family in orchardgrass.

12864_2025_11241_MOESM6_ESM.jpg (1.5MB, jpg)

Supplementary Material 6: Supplementary Figure S1 The chromosome location of the ARF gene family in orchardgrass.

12864_2025_11241_MOESM7_ESM.jpg (5.1MB, jpg)

Supplementary Material 7: Supplementary Figure S2 The cis-acting regulatory elements of DgARF gene promoters (2000 bp) in orchardgrass. (a) The distribution of cis-acting regulatory elements of DgARFs. (b) The number of cis-acting regulatory elements of DgARFs.

Data Availability Statement

The genome data and total protein sequences of orchardgrass were obtained from NCBI BioProject database (https//www.ncbi.nlm.nih.gov/bioproject) using the accession number PRJNA471014. The ARF protein sequences for Arabidopsis thaliana, Oryza sativa, Triticum aestivum, Zea mays, Raphanus sativus, Solanum tuberosum, Solanum lycopersicum, Vigna radiata, Medicago truncatula, Malus domestica, and Prunus mume were sourced from the Plant transcription factor database (https://planttfdb.gao-lab.org/). All data generated or analyzed during this study are included in this article and can be found in the Additional file.

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