Genome-wide identification and expression pattern of the phytochrome-interacting factors (PIFs) family in three alfalfa varieties

Abstract

Background

The Phytochrome-interacting factors (PIFs) are members of the basic helix-loop-helix (bHLH) transcription factor family and play essential roles in plant growth, development, and stress response. While the PIF gene family has been extensively studied in various plant species, there is limited information available regarding their presence in high-quality perennial legume alfalfa (Medicago sativa).

Results

In this study, we identified 29, 9, and 27 genes in Xinjiang Daye, Zhongmu No. 1, and Zhongmu No. 4 alfalfa varieties, respectively. These genes are unevenly distributed on 5 chromosomes of Xinjiang Daye, 3 chromosomes of Zhongmu No. 1, and 7 chromosomes of Zhongmu No. 4, with 2 genes not located on any chromosome. Phylogenetic analysis revealed that the MsPIFs genes clustered into five different branches (PIF I to PIF V), with members within the same subfamily sharing conserved motifs and displaying similar exon-intron distribution patterns. Gene duplication analysis indicated that segmental duplications facilitated the expansion and evolution of the alfalfa PIF gene family. Functional cis-element analysis of the MsPIFs genes promoter regions identified elements related to light, hormones, development, and response to abiotic stresses. The expression levels of the majority of MsPIF family genes significantly varied under drought, high temperature, and combined stress conditions, notably showing pronounced responses in genes MsPIF4, MsPIF6, and MsPIF9 to these stressors.

Conclusion

We systematically identified and classified the members of the MsPIFs gene family, analyzing their chromosomal locations and genetic structure. These MsPIFs are crucial in alfalfa’s responses to drought and high-temperature stress.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12864-025-11826-0.

Introduction

Alfalfa (Medicago sativa L.) is a vital leguminous forage crop, that predominantly cultivated in arid and semi-arid regions [1]. It is renowned as the 'Queen of Forages' for its high protein content, strong adaptability, and excellent palatability [2]. In recent years, with the transformation and advancement of the livestock industry, there has been a growing annual demand for high yield and outstanding quality forage, leading to a sharp increase in the demand for alfalfa. However, as global warming and increasing water scarcity, the production and utilization of alfalfa are severely constrained [3, 4]. Slama et al. [5] studied eight alfalfa varieties and observed a 55–75% reduction in biomass under the condition of water shortage. Under high-temperature stress, alfalfa exhibits a significant decrease in chlorophyll content and chlorophyll fluorescence parameters (Fv / Fm), along with a down-regulation of crucial photosynthesis-related proteins. The levels of malondialdehyde (MDA) and electrolyte leakage (EL) in alfalfa show an increasing trend [6]. Furthermore, it is indicated that the simultaneous occurrence of drought and high temperature has a far greater adverse impact on the yield and quality of alfalfa than when they appear individually [7]. Therefore, drought and high temperature have emerged as primary limiting elements influencing plant growth and development as well as human productivity.

Transcription factors are the predominant gene regulatory entities in the genomes of multicellular organisms. They modulate gene expression universally by binding to specific DNA sequences to either activate or inhibit the expression of target genes [8–10]. The phytochrome-interacting factors (PIFs) belong to 15th subfamily of the basic-helix-loop-helix (bHLH) transcription factor superfamily and serves as a key transcriptional regulatory factor that governs responses to light and environmental stimuli [11, 12]. Members of this subfamily possess a conserved active phytochrome B-binding (APB) domain at the N-terminus and / or an active phytochrome A-binding (APA) domain which enables specific interactions with phytochrome B (phyB) and / or phytochrome A (phyA) for the regulation of photomorphogenesis [13, 14]. Interestingly, PIFs play leading roles not only in light signal transduction but also in integrating environmental cues with internal signals such as hormones and the biological clock. This integration is crucial for regulating various aspects of plant growth and development, including seed germination, flowering, hypocotyl elongation, shade avoidance, and chloroplast development [15–17]. Currently, it has been demonstrated that PIFs can participate in regulating the biosynthesis and signaling pathways of various plant hormones such as abscisic acid (ABA), gibberellins (GA), brassinosteroids (BRs), jasmonic acid (JA), auxins, and ethylene (ETH), thereby synergistically controlling plant growth and development [18–21]. Additionally, AtPIF1/PIL5 functions as a negative regulator in the process of chlorophyll biosynthesis and seed germination under low-light conditions [22, 23]. PIF3 negatively regulates chloroplast development and chlorophyll synthesis [24, 25], while PIF4 positively regulates the phytochrome-mediated plant shade avoidance response and flowering time [26].

Simultaneously, PIF transcription factors serve as central regulators of plant stress adaptation, orchestrating the integration of light, temperature, and hormonal signals to regulate gene expression networks under abiotic stress conditions. AtPIF4 enhances plant thermotolerance by transcriptionally repressing SPEECHLESS (AtSPCH) expression, thereby regulating stomatal closure in leaves under high-temperature conditions in Arabidopsis thaliana [27]. The transcription factor TEOSINTE BRANCHED 1/CYCLOIDEA/PCF 5 (AtTCP5) catalyzed hypocotyl elongation, augmented AtPIF4 gene activity, and interacted directly with the AtPIF4 gene promoter, consequently stimulating AtPIF4 gene expression during heat stress [28]. PIFs orchestrate a network of histone modifications (H2A.Z eviction, acetylation), chromatin remodeling (INO80 complex), and transcriptional memory to enhance plant resilience against high temperatures. These mechanisms ensure rapid and adaptive responses to thermal stress [29]. Under high-temperature conditions, PIF4 is stabilized by HEMERA (HMR) protein, which activates genes such as YUCCA8 (YUC8), Indole-3-acetic acid inducible 19 (IAA19) to promote IAA biosynthesis, thereby regulating thermomorphogenesis [30]. In Arabidopsis thaliana, AtPIF3 interacts with the C-repeat binding factor (CBF) to enhance the stability of the PIF3 protein, consequently regulating the expression of growth-related genes through the phyB-PIF signaling pathway to improve plant cold tolerance [31]. SIPIF4 may bind to the G-box in the promoter of Gibberellic Acid Insensitive 4 (SIGAI4), which encodes a DELLA protein, thereby enhancing low-temperature tolerance in tomato (Solanum Iycopersicum L.). Additionally, SIPIF4 promotes the biosynthesis of JA and ABA, while suppressing GA biosynthesis under cold stress [32]. Furthermore, Gao et al. [33] found that the ZmPIF1 enhances drought resistance and increases yield by inducing stomatal closure in maize (Zea mays L.). Research has indicated that MfPIF1 upregulates the expression of ABA-responsive genes NCED3, Δ-1-pyrroline-5-carboxylate synthetase (P5CS), and RD29A post-drought, enhancing ABA biosynthesis in Myrothamnus flabellifolia. Moreover, MfPIF1 elevates osmotic regulatory substance levels in plants, boosts antioxidant enzyme activities, reduces MDA and reactive oxygen species (ROS) accumulation, and mitigates drought-induced damage [34]. Therefore, the PIFs play a pivotal regulatory role in defensing adversity stress.

Nowadays, the PIFs gene family has been identified in multiple species. Members of the PIFs gene family vary among different species, exhibiting diverse biological functions. However, the previously reported identification of PIF gene families in crops such as Arabidopsis thaliana [13], Oryza sativa [35], Glycine max [36], and Zea mays [37] has been based on individual genomes, resulting in incomplete characterization of family members. Furthermore, the PIF gene family members in Medicago sativa have not been well characterized, and it remains unclear whether PIF family genes are involved in responding to drought and heat stress in alfalfa. Therefore, this study utilized bioinformatics methods to identify the PIFs gene family in alfalfa, based on publicly available whole-genome data. The research encompassed the analysis of their physicochemical properties, conserved motifs, chromosomal mapping, phylogenetic tree, promoter cis-regulatory elements, and real-time fluorescence quantitative PCR to assess the expression of PIFs genes under drought and high-temperature conditions. This comprehensive approach aims to lay a theoretical foundation for further elucidating the functional roles of the PIFs genes in alfalfa. This study utilized bioinformatics methods to identify the PIFs gene family in alfalfa based on the whole-genome data of Xinjiang Daye [38], Zhongmu No. 1 [39], and Zhongmu No. 4 [40] have been published. Additionally, RT-qPCR was employed to assess the expression of PIFs genes under drought and high-temperature stress. The analysis of this research can serve as a theoretical basis for further elucidating the functional roles of PIFs genes in alfalfa and cloning of MsPIFs genes.

Materials and methods

Identification and characterization of the PIFs gene family in alfalfa

The PIF genes in Arabidopsis thaliana, rice (Oryza sativa), soybean (Glycine max), and Trifolium repens were used as references. The genomic and annotation data for Arabidopsis thaliana were obtained from the TAIR database (https://www.arabidopsis.org/), while nucleotide sequences for rice, soybean, and Trifolium repens were sourced from NCBI (https://www.ncbi.nlm.nih.gov/gene). The whole genome data and annotation files of cultivated alfalfa used in this study were sourced from the MODMS (https://modms.lzu.edu.cn/) website. The MsPIFs genes were searched using the following approach: a local BLAST database was established utilizing downloaded Medicago sativa genome data. Reference species gene sequences, already downloaded, served as query sequences to explore homologous genes in the aforementioned database; corresponding gene sequences were then extracted based on the obtained homologous gene IDs. Identification of genes containing the HLH domain were identified through NCBI Batch CD-Search (https://www.ncbi.nlm.nih.gov) analysis, and the final genes selected for further analysis were obtained by eliminating redundancy (https://web.expasy.org/decrease_redundancy).

Subsequently, the protein length, molecular weight (Mw), theoretical isoelectric point (pI), and grand average of hydropathicity (GRAVY) of PIFs genes were investigated using the ProtParam online tool available on the ExPASy (http://web.expasy.org/protparam/). The subcellular localization (Sl) of PIFs proteins were predicted using the online tool WoLF PSORT (https://wolfpsort.hgc.jp/). All relevant details about the PIFs genes are summarized in Table 1.

Table 1.

Identification of the characteristics of PIFs genes in alfalfa

Gene ID	Gene Name	AA	Mw (kDa)	pI	GRAVY	Sl
MS.gene038012.t1	MsPIF1-X1-1	538	59.98	5.80	-0.782	Nucl
MS.gene059011.t1	MsPIF1-X1-2	531	59.00	5.89	-0.774	Nucl
MS.gene97692.t1	MsPIF1-X1-3	546	60.57	5.93	-0.756	Nucl
MS.gene24034.t1	MsPIF1-X1-4	546	60.57	5.80	-0.773	Nucl
MS.gene005322.t1	MsPIF2-X1-1	202	22.53	11.05	-0.814	Nucl
MS.gene40778.t1	MsPIF2-X1-2	201	22.41	11.05	-0.766	Nucl
MS.gene21011.t1	MsPIF2-X1-3	202	22.53	11.05	-0.814	Nucl
MS.gene21033.t1	MsPIF2-X1-4	201	22.41	11.05	-0.766	Nucl
MS.gene005375.t1	MsPIF3-X1-1	719	76.90	6.20	-0.661	Nucl
MS.gene22936.t1	MsPIF3-X1-2	719	76.82	6.35	-0.678	Nucl
MS.gene20962.t1	MsPIF3-X1-3	719	76.80	6.35	-0.678	Nucl
MS.gene21084.t1	MsPIF3-X1-4	719	76.82	6.35	-0.679	Nucl
MS.gene46957.t1	MsPIF4-X3-1	527	58.72	6.89	-0.720	Nucl
MS.gene22222.t1	MsPIF4-X3-2	515	57.38	6.84	-0.738	Nucl
MS.gene019150.t1	MsPIF5-X3-1	338	37.10	9.30	-0.759	Nucl
MS.gene015170.t1	MsPIF6-X5-1	347	37.87	6.07	-0.629	Nucl
MS.gene041064.t1	MsPIF6-X5-2	347	37.92	6.07	-0.645	Nucl
MS.gene047611.t1	MsPIF6-X5-3	347	38.11	6.01	-0.659	Nucl
MS.gene010332.t1	MsPIF6-X5-4	347	37.86	6.21	-0.615	Nucl
MS.gene96615.t1	MsPIF7-X6-1	302	34.21	4.96	-0.398	Cyto
MS.gene36203.t1	MsPIF8-X7-1	435	46.70	9.30	-0.506	Nucl
MS.gene010774.t1	MsPIF9-X7-1	492	52.93	8.41	-0.559	Nucl
MS.gene38309.t1	MsPIF9-X7-2	492	52.99	8.60	-0.559	Nucl
MS.gene93265.t1	MsPIF9-X7-3	492	53.01	8.62	-0.564	Nucl
MS.gene09877.t1	MsPIF10-X7-1	488	54.23	5.57	-0.670	Nucl
MS.gene018583.t1	MsPIF11-X7-1	543	60.34	5.35	-0.540	Nucl
MS.gene061863.t1	MsPIF12-X7-1	494	55.02	5.57	-0.638	Nucl
MS.gene64151.t1	MsPIF12-X7-2	494	55.02	5.57	-0.638	Nucl
MS.gene061862.t1	MsPIF12-X7-3	492	54.73	5.41	-0.623	Nucl
MsG0180000277.01.T01	MsPIF1-Z11-1	239	26.48	6.29	-0.564	Nucl
MsG0180003748.01.T01	MsPIF2-Z11-2	539	59.88	5.89	-0.678	Nucl
MsG0180004666.01.T01	MsPIF3-Z11-1	201	22.41	11.05	-0.766	Nucl
MsG0180004703.01.T01	MsPIF4-Z11-1	715	76.39	6.43	-0.671	Nucl
MsG0180004703.01.T02	MsPIF4-Z11-2	715	76.39	6.43	-0.671	Nucl
MsG0380013499.01.T03	MsPIF5-Z13-1	221	24.43	9.46	-0.885	Nucl
MsG0780038030.01.T01	MsPIF6-Z17-1	492	52.98	8.41	-0.570	Nucl
MsG0780038108.01.T01	MsPIF6-Z17-2	492	53.06	8.76	-0.567	Nucl
MsG0780040838.01.T01	MsPIF7-Z17-1	474	52.63	5.33	-0.584	Golg
Msa0026930-mRNA-1	MsPIF1-Z41-1	542	60.36	5.80	-0.770	Nucl
Msa0074760-mRNA-1	MsPIF1-Z41-2	546	60.57	5.93	-0.762	Nucl
Msa0074780-mRNA-1	MsPIF1-Z41-3	542	60.36	5.80	-0.770	Nucl
Msa1145430-mRNA-1	MsPIF1-Z47-4	546	60.57	5.80	-0.773	Nucl
Msa0036780-mRNA-1	MsPIF2-Z41-1	719	76.82	6.35	-0.678	Nucl
Msa0132010-mRNA-1	MsPIF2-Z41-2	719	76.81	6.35	-0.689	Nucl
Msa0081010-mRNA-1	MsPIF2-Z41-3	719	76.81	6.35	-0.689	Nucl
Msa0269960-mRNA-1	MsPIF2-Z42-4	719	76.81	6.35	-0.688	Nucl
Msa0051890-mRNA-1	MsPIF3-Z41-1	267	29.39	5.78	-0.714	Nucl
Msa0096880-mRNA-1	MsPIF3-Z41-2	267	29.39	5.78	-0.714	Nucl
Msa0144870-mRNA-1	MsPIF3-Z41-3	267	29.39	5.78	-0.714	Nucl
Msa1441050-mRNA-1	MsPIF3-Z41-4	267	29.39	5.78	-0.714	Nucl
Msa0185930-mRNA-1	MsPIF4-Z42-1	201	22.41	11.05	-0.766	Nucl
Msa0342990-mRNA-1	MsPIF5-Z43-1	347	37.86	5.94	-0.634	Nucl
Msa0714770-mRNA-1	MsPIF5-Z45-2	347	37.87	6.07	-0.629	Nucl
Msa0756840-mRNA-1	MsPIF5-Z45-3	347	37.89	6.07	-0.637	Nucl
Msa0836170-mRNA-1	MsPIF5-Z45-4	347	37.85	5.94	-0.613	Nucl
Msa0358710-mRNA-1	MsPIF6-Z43-1	524	58.47	7.22	-0.757	Nucl
Msa0404870-mRNA-1	MsPIF6-Z43-2	524	58.47	7.22	-0.757	Nucl
Msa0450880-mRNA-1	MsPIF6-Z43-3	524	58.41	7.20	-0.763	Nucl
Msa0482580-mRNA-1	MsPIF6-Z43-4	524	58.47	7.22	-0.757	Nucl
Msa1016450-mRNA-1	MsPIF7-Z47-1	492	52.98	8.41	-0.570	Nucl
Msa1057940-mRNA-1	MsPIF7-Z47-2	492	52.95	8.62	-0.559	Nucl
Msa1042420-mRNA-1	MsPIF8-Z47-1	453	50.32	5.57	-0.657	Nucl
Msa1078210-mRNA-1	MsPIF9-Z47-1	549	61.11	5.50	-0.552	Nucl
Msa1123440-mRNA-1	MsPIF9-Z47-2	540	59.97	5.49	-0.541	Nucl
Msa1451200-mRNA-1	MsPIF9-Z47-3	549	61.14	5.50	-0.547	Nucl

AA Number of amino acids, Mw Molecular weight, pI Theoretical PI, GRAVY Grand average of hydropathicity, Sl Subcellular localization, Nucl Nucleus, Cyto Cytoplasm, Golg Golgi apparatus

Phylogenetic analysis within PIFs genes

The PIFs protein sequences of alfalfa were aligned with those of Arabidopsis, Oryza sativa, Glycine max, and Trifolium repens using TBtools [41]. Following the alignment, a neighbor-joining method was applied in MEGA 7.0 to build a phylogenetic tree for the PIFs members, with the bootstrap value set at 1000 and the remaining parameters left at default settings [42].

Conserved motif and gene structure analysis of MsPIFs

The prediction of conserved motifs in the PIFs proteins of alfalfa was conducted using MEME program (https://meme-suite.org/meme/tools/meme), with default parameters and maximum number of motifs set at 15, followed by visualization through the TBtools. Moreover, TBtools [41] was utilized to showcase the gene structure and detect the exon/intron boundaries.

Examination of chromosome distribution and gene duplication

Chromosome positions of the PIFs gene family members were predicted in TBtools. Subsequently, the collinearity of PIFs genes in Medicago sativa cultivars Xinjiang Daye, Zhongmu No. 1, and Zhongmu No. 4 was analyzed using MCScanX [43], and the results were visualized using TBtools.

Analysis of Cis-acting element in the PIFs genes family of alfalfa

The TBtools was utilized to extract a 2000 bp sequence upstream of the PIFs transcription start site in alfalfa as the promoter sequence. This sequence was submitted to PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) for cis-elements analysis, and the results were visualized with TBtools.

Naming of the PIFs genes in alfalfa

The nomenclature of PIFs family members comprises four parts: the first is the abbreviation of the species name, where Ms. represents Medicago sativa L; the second part includes the abbreviation of the Phytochrome Interacting Factor (PIF) and the sequence in which different members appear on the chromosome; the third part consists of abbreviations of different alfalfa varieties and their chromosome numbering sequence, namely X for Xinjiang Daye, Z1 for Zhongmu No. 1, and Z4 for Zhongmu No. 4; the fourth part indicates the ordering of homologous genes on different chromosomes, determined by the order in which homologous chromosomes appear in the genome. Thus, the gene named MS. gene038012.t1 is denoted as MsPIF1-X1-1, signifying its membership in the alfalfa PIFs gene family, and the first homologous chromosome and the first gene of Xinjiang Daye.

Plants cultivation and abiotic stress treatments

The experimental materials consisted of the cultivated alfalfa varieties 'Gannong No. 7, ''Zhongmu No. 1,'' Zhongmu No. 4,' and 'Xinjiang Daye'. The seeds were respectively provided by the Pratacultural College of Gansu Agricultural University, the Institute of Animal Sciences of Chinese Academy of Agricultural Sciences, and the National Livestock Husbandry Station, Ministry of Agriculture and Rural Affairs of The People’s Republic of China.

Seeds with plump grains were sown in pots (diameter: 15 cm; height: 14 cm) filled with nutrient soil and placed in an artificial climate chamber with a light intensity of 2000 lx, a 16-hour light period, a temperature of 25 °C, and a humidity of 70%; during the 8-hour dark period, the temperature was 20 °C, and the humidity was 60%. Following germination, uniform alfalfa seedlings were transplanted into hydroponic boxes (12 cm × 8 cm × 11 cm, length × width × height) with 6 seedlings per box, and were further nurtured using Hoagland nutrient solution. The nutrient solution was replenished every 3 days throughout the cultivation period. Five-weeks-old seedlings were subjected to drought and high-temperature stress treatments.

The pre-cultured seedlings were categorized into three groups: the drought treatment group (Drought), the high-temperature treatment group (Heat), and the combined drought and high-temperature treatment group (Drought + Heat). The drought treatment group underwent simulated drought stress using a 0.1% sorbitol solution. The high-temperature treatment group experienced 38 °C for 14 h/day and 25 °C for 10 h/day. The drought and high-temperature combined treatment group is subjected to the same temperature as the high-temperature treatment group on the basis of 0.1% sorbitol treatment. The nutrient solution was renewed daily throughout the stress period. Leaves of the seedlings were gathered at 0, 6, 12, 24, and 72 h after treatment, then immediately frozen and stored at -80 °C until use. Four biological replicates were utilized for each of the mentioned time points.

RNA isolation and RT-qPCR verification

Total RNA samples were extracted using the RNAprep pure Plant Kit (Tiangen, Beijing, China). 1 µg of RNA was utilized for the synthesis of first-strand cDNA with the PrimerScript™ RT Reagent Kit containing gDNA Eraser (TaKaRa, Otsu, Japan). Primer design was conducted through Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/). RT-qPCR analysis was carried out using the SYBR Green I Master reaction system and the LightCycler 96 PCR instrument (Roche, Rotkreuz, Switzerland). The Msactin gene was utilized as the internal reference in each reaction. The reaction system and PCR settings were determined based on the kit guidelines, and the relative gene expression levels were determined using the 2^−ΔΔCt method [44]. Data analysis was conducted using SPSS 25. The data were presented as the mean ± standard error of three biological replicates with two technical replicates and visualized using GraphPad Prism 9.0.0. The RT-qPCR validation primers utilized in this study are enumerated in Table S1.

Results

Identification and analysis of PIFs genes in Medicago sativa

A total of 65 MsPIFs genes, including allelic genes, were identified in Xinjiang Daye (29), Zhongmu No. 1 (9), and Zhongmu No. 4 (27) (Table 1). Determination physicochemical property on all members of MsPIFs, the number of amino acids (AA) ranged from 201 (MsPIF2-X1-2, MsPIF2-X1-4, MsPIF4-Z42-1) to 719 (MsPIF3-X1-1 / 2 / 3 / 4, MsPIF2-Z41-1 / 2 / 3, MsPIF2-Z42-4) (Table 1). The predicted molecular weight (Mw) of MsPIFs between 22.41 and 76.90 kDa and the theoretical pI values (pI) ranged from 4.96 to 11.05. Among the identified 65 PIFs genes, forty-five have a pI value less than 7.00, classifying them as acidic proteins, while the rest exhibit alkaline properties. The grand average of hydropathicity (GRAVY) from − 0.885 (MsPIF5-Z13-1) to -0.398 (MsPIF7-X6-1), indicating that all PIFs proteins are hydrophilic. Furthermore, subcellular localization prediction reveals that 97% of MsPIFs proteins localized in the nucleus, with the remaining two divided between the cytoplasm (MsPIF7-X6-1) and the Golgi apparatus (MsPIF7-Z17-1) (Table 1). These results affirm substantial distinctions in sequence and protein characteristics among the 65 MsPIFs proteins.

Phylogenetic analysis of MsPIFs genes

Figure 1 illustrates the utilization of MEGA 7.0 software for conducting multiple sequence alignment and phylogenetic tree construction of the PIFs genes family in Medicago sativa (Xinjiang Daye, Zhongmu No. 1, Zhongmu No. 4), M. sativa spp. caerulea, Medicago truncatula, Arabidopsis (Table S3), rice, soybean, and Trifolium repens (Table S4). Based on the similarity of their amino acid sequences, the phylogenetic tree divides PIF family members into 5 classes and 7 subfamilies, namely: PIF I, PIF II (a, b), PIF IIII (a, b), PIF IV, and PIF V. Apart from the PIF I subfamily, MsPIFs genes are distributed in various subfamilies. The tree includes a total of 122 PIFs proteins, with the PIF IIa subfamily having the highest number of members (26 PIFs), encompassing 9 MsPIFs, 11 OsPIFs, 3 GmPIFs, 1 MtPIFs, and 2 AtPIFs. In contrast, the PIF I subfamily has the lowest proteins, featuring only 7 AtPIFs. The PIF II b subfamily comprises a total of 25 members, with 21 being MsPIFs. The PIF III a subfamily exists 20 PIFs genes and contains 14 MsPIFs members. There are 22 PIFs in the PIF IV subfamily, including 13 MsPIFs, and none are AtPIF category. Moreover, the PIF III b and PIF V subfamilies each consist of 11 PIF members, and the MsPIFs genes are present in 9 and 8 copies, respectively.

Fig. 1.

Phylogenetic tree based on the relationships among Medicago sativa (Ms) and Medicago sativa spp. Caerulea (Ms), Medicago truncatula (Mt), Arabidopsis thaliana (At), Oryza sativa (Os), Glycine max (Gm), and Trifolium repens (Tr). The 7 subfamilies are distinguished by distinct colors

Detection of conserved motif and gene structure

In order to deepen the understanding of the molecular structure and functional characteristics of MsPIFs, we analyzed the motifs and gene structure of its family members. Through motif analysis, a total of 15 different conserved motifs of the MsPIFs proteins were identified by the MEME website and designated as motif 1 to 15 (Fig. 2). Some motifs are conserved among MsPIFs members, while others are unique to only a few MsPIFs members. For example, motif 1 and 2 were identified in 59 MsPIFs proteins, with the exceptions of MsPIF2-X1-4, MsPIF2-X1-2, MsPIF2-X1-1, MsPIF2-X1-3, MsPIF7-X6-1, MsPIF5-Z13-1, MsPIF2-Z11-2, MsPIF4-Z11-2, MsPIF4-Z11-1, MsPIF4-Z42-1, and MsPIF3-Z11-1, which exclusively contain motif 1 or 2. Additionally, six MsPIFs only including motif 2 and motif 10 (Fig. 2B). Out of the 65 MsPIFs proteins, 59 MsPIFs (91%) contain four or more conserved motifs. Furthermore, 17 genes (MsPIF1-Z41-2, MsPIF1-X1-2, MsPIF1-Z41-3, MsPIF1-X1-1, MsPIF1-Z41-1, MsPIF1-Z47-4, MsPIF1-X1-4, MsPIF1-X1-3, MsPIF10-X7-1, MsPIF12-X7-2, MsPIF11-X7-1, MsPIF12-X7-1, MsPIF9-Z47-3, MsPIF9-Z47-2, MsPIF12-X7-3, MsPIF9-Z47-1, and MsPIF7-Z17-1) carry motifs 1 through 15. Genes with closer phylogenetic relationships exhibit more similar types, quantities, and positions of motif distribution, displaying a high level of conservation, such that PIF III b subfamily all members contained motifs 1–8, 11 and 12 except MsPIF8-X7-1 (Fig. 2B).

Fig. 2.

Analysis of the phylogenetic relationships, conserved motifs, and gene structures of MsPIFs genes. A Phylogenetic tree of 65 MsPIFs proteins. An unrooted neighbor-joining phylogenetic tree was constructed using the full-length amino acid sequences of the 65 MsPIFs proteins with MEGA7.0, and 1000 bootstrap replicates were performed. B Composition of the conserved motifs in MsPIFs proteins. Motifs are labeled from 1 to 15 and are represented by different colored boxes. C The exon-intron structure of MsPIFs genes. Untranslated 5’- and 3’- regions are indicated by yellow boxes; exons are represented by green boxes; introns are depicted by black lines

The gene structure analysis revealed that the number of CDS ranged from 1 to 10, with over half of the genes containing UTR regions (Fig. 2C). The results suggest that genes within the clustering subfamily, sharing closer phylogenetic relationships, demonstrate more uniform exon arrangements and consistent intron lengths. The PIF II b and PIF III b subfamilies comprise 6–10 and 3–6 exons, respectively, with relatively longer introns. Conversely, the PIF II a, PIF III a, and PIF IV subfamilies consist of 5–7 exons, with shorter introns. In contrast, the PIF V subfamily genes (MsPIF2-X1-1 / 2 / 3 / 4, MsPIF4-Z42-1, and MsPIF3-Z11-1) exhibit quite conservation, consisting of only 1 exon and the shortest gene lengths.

Chromosomal localization and collinearity analysis of the MsPIFs genes

The chromosomal localization analysis of the MsPIFs genes family members was performed using the TBtools software. The results indicated that 65 MsPIFs genes unevenly distributed on five chromosomes (chr) of Xinjiang Daye, three chromosomes of Zhongmu No. 1, and seven chromosomes of Zhongmu No. 4, with two genes (MsPIF3-Z41-4, MsPIF9-Z47-3) are not located on any chromosome (Fig. 3). The position that genes belonging to the same subfamily MsPIFs are almost identically in Xinjiang Daye, Zhongmu No. 1, and Zhongmu No. 4. For example, the chromosomal and relative locations of MsPIF4-X3-2, MsPIF5-Z13-1, and MsPIF6-Z43-3 remain consistently stable, highlighting a marked level of conservation in the organization of MsPIFs genes. Furthermore, the majority of chromosomes harbored 1 or 2 MsPIFs genes, with the highest distribution observed on chr 7.4 in Xinjiang Daye and Chr 1 in Zhongmu No. 1, each containing 5 MsPIFs genes, followed by chr 1.2 of Xinjiang Daye and chr 1_2 of Zhongmu No. 4, each possesses 4 MsPIFs genes. Three MsPIFs genes were detected on chr 1.1, chr 1.3, and Chr 7, respectively.

Fig. 3.

Chromosomal location and gene duplication of MsPIFs in Xinjiang Daye (orange cylinder), Zhongmu No. 1 (green cylinder), and Zhongmu No. 4 (blue cylinder). The tandem duplicated genes are highlighted in red-colored font and the chromosomes without MsPIFs genes were eliminated

Gene duplication is essential for the generation of new genes and functions, with segmental and tandem duplications serving as important driving forces for the expansion of gene families. Repeat analysis showed that 8 MsPIFs genes (12.31%) were identified as tandem repeat genes. Including two pairs of independent tandem duplicated genes are located on Chr 1 of Zhongmu No. 1 and chr 1_2 of Zhongmu No. 4 (MsPIF4-Z11-1 / MsPIF4-Z11-2 and MsPIF1-Z41-2 / MsPIF1-Z41-3) and one group of four tandem duplicated genes (MsPIF12-X7-1, MsPIF12-X7-2, MsPIF11-X7-1, and MsPIF12-X7-3) located on chr 7.4 of Xinjiang Daye (Fig. 3). Moreover, homology analysis of MsPIFs genes among three different alfalfa varieties revealed that of numerous genes were involved in segmental duplication events, particularly between Xinjiang Daye and Zhongmu No. 4 (Fig. 4). 38, 2, and 18 sets tandem repeat genes were respectively identified in Xinjiang Daye, Zhongmu No. 1, and Zhongmu No. 4. Additionally, 37, 80, and 23 gene pairs involved in fragment duplicated events were identified Xinjiang Daye vs. Zhongmu No. 1, Xinjiang Daye vs. Zhongmu No. 4, and Zhongmu No. 1 vs. Zhongmu No. 4. These results imply that both tandem repeats and segmental repeats jointly promoted the expansion of the MsPIFs family.

Fig. 4.

Homologous MsPIF genes of X vs. Z1 (A), X vs. Z4 (B), and Z1 vs. Z4 (C). The blue blocks represent the chromosomes; gray lines in the background represent collinear blocks among the genomes of three different varieties; green and red lines highlight the homologous MsPIFs genes pairs in Xinjiang Daye, Zhongmu No. 1, and Zhongmu No. 4; yellow lines highlight homologous MsPIFs genes pairs in different varieties

The collinearity analysis among three alfalfa cultivars (Xinjiang Daye, Zhongmu No. 1, and Zhongmu No. 4) and related species (Arabidopsis thaliana, Oryza sativa, and Medicago truncatula) revealed distinct evolutionary and genomic conservation patterns. The alfalfa cultivars exhibited the strongest collinearity with Medicago truncatula, characterized by large continuous syntenic blocks, indicating minimal genomic rearrangements and high conservation within the Medicago genus. The collinearity with Arabidopsis thaliana was fragmented, with short syntenic segments. In Arabidopsis thaliana, six and five PIF family genes exhibited high homology with PIF family genes in Xinjiang Daye and Zhongmu No. 4, respectively, while no orthologous genes were detected between Zhongmu No. 1 and Arabidopsis thaliana (Fig. 5A-C). In addition, the collinearity with Oryza sativa was sparse and highly fragmented, reflecting the deep evolutionary split between monocots and eudicots. Compared with Zhongmu No. 1 and Zhongmu No. 4, the highest number of orthologous gene pairs in the PIF family was detected between Xinjiang Daye and Oryza sativa. However, when compared with Arabidopsis thaliana and Medicago truncatula, Oryza sativa exhibited the lowest homology with Medicago sativa (Fig. 5). Notably, Xinjiang Daye and Zhongmu No. 1 showed slightly higher collinearity with Medicago truncatula, suggesting its closer genetic proximity to Medicago truncatula (Fig. 5G-I). These results underscore the dual influence of conserved genomic architecture within closely related species and selective gene retention across distant taxa during alfalfa evolution.

Fig. 5.

Collinearity analysis among Arabidopsis thaliana, Oryza sativa, Medicago truncatula, and Medicago sativa. A-C Collinearity analysis between Arabidopsis thaliana and Xinjiang Daye (A), Zhongmu No. 1 (B), and Zhongmu No. 4 (C). D-F Collinearity analysis between Oryza sativa and Xinjiang Daye (D), Zhongmu No. 1 (E), Zhongmu No. 4 (F). G-I Collinearity analysis between Medicago truncatula and Xinjiang Daye (G), Zhongmu No. 1 (H), Zhongmu No. 4 (I). Gray lines in the background represent collinear blocks between Medicago sativa and other plant genomes; Red highlighted lines indicate homologous gene pairs of the PIF gene family between two genomes. At, Os, Mt, XJDY, ZM1, and ZM4 represent Arabidopsis thaliana, Oryza sativa, Medicago truncatula, Xinjiang Daye, Zhongmu No. 1, and Zhongmu No. 4, respectively

Analysis of cis-regulatory elements in the promoter of MsPIFs genes

To further investigate the potential biological functions of the MsPIFs gene, we analyzed the cis-acting elements within the 2000 bp upstream promoter region of MsPIFs using the online PlantCARE database and visualized the results using TBtools. Five classes of cis-acting elements totaling 47 have been identified, encompassing hormone responsive (10), development responsive (8), anaerobic responsive (2), stress responsive (5), and light responsive (22). Of these, light, hormone, and development responsive elements are the most prevalent, highlighting the significant roles of the alfalfa PIF gene family in regulating plant growth and stress resistance (Fig. 5). Specifically, hormone response elements encompass components associated with auxin responsive (TGA-element, TGA-box), related to gibberellin-responsiveness (TATC-box, GARE-motif, P-box), related to MeJA-responsiveness (CGTCA-motif, TGACG-motif), related to abscisic acid responsiveness (ABRE), related to salicylic acid responsiveness (TCA-element), and zein metabolism regulation (O2-site). Development responsive elements include flavonoid biosynthetic genes regulation (MBSI), endosperm expression (GCN4_motif, AACA_motif), meristem expression (CAT-box). The cis-regulatory elements involved in abiotic stress can be classified into drought-inducibility (MBS), AT-rich element, MYBHv1 binding site (CCAAT-box), low-temperature responsiveness (LTR), and defense and stress responsiveness (TC-rich repeats). The anaerobic induction (ARE) and anoxic specific inducibility (GC-motif) are linked to anaerobic response. The light responsive cis-acting elements contain ATCT-motif, Box 4, GT1-motif, Sp1, Box II, chs-CMA1a, chs-CMA2a, GA-motif, Gap-box, GATA-motif, GATT-motif, GTGGC-motif, I-box, LAMP-element, TCCC-motif, TCT-motif, AT1-motif, ACE, G-Box, MRE, AE-box, and circadian control. Among these elements, approximately 80% of genes contain the GT1-motif and G-box, while nearly 90% of genes harbor the Box-4 element. Moreover, the figure illustrates that every gene harbors at least 10 cis-elements, with the quantity of identical cis-elements in distinct genes varying between 1 and 9. For instance, MsPIF7-Z47-2 and MsPIF1-X1-1 contain 9 and 7 Box4 cis-elements, respectively, and MsPIF9-X7-1 and MsPIF9-X7-2 consist of 5 and 9 MRE light-responsive elements, respectively.

Analysis of MsPIFs genes expression under abiotic stress

To elucidate the modulation of alfalfa PIF gene expression under diverse abiotic stresses including drought, heat, and their combination, qRT-PCR technology was employed to analyze the transcription levels of MsPIFs genes with 4 biological replicates. To effectively showcase the gene expression levels of four varieties of alfalfa in a bar chart, we renamed the names of the previously identified genes and incorporated the relevant data into Table S2. A total of 17 non-redundant MsPIF family members (MsPIF1–MsPIF17) were identified through the alfalfa genomes of Xinjiang Daye, Zhongmu No. 1, and Zhongmu No. 4 (Table S2). The relative expression level of MsPIF10 was not quantified due to potential inaccuracies caused by nonspecific primers. In general, the relative expression levels of most PIF genes exhibit the most significant fluctuations at 12 h after the stress treatment, with the gene expression in Gan Nong No. 7 surpassing those in the other three varieties. For instance, following 12 h of drought stress, the relative expression levels of MsPIF4 in Gannong No. 7 are 102-fold, 29-fold, and 151-fold higher than those of Zhongmu No. 1, Zhongmu No. 4, and Xinjiang Daye, respectively (Figs. 7 and 8). Under drought conditions, the relative expression levels of various genes in Gannong No. 7 peaked at 12 h treatment and then decreased. In contrast to the 0 h, nine MsPIFs genes expression (MsPIF1 /4 /6 /7 /9 /11 / 16) in Zhongmu No. 1 demonstrated elevated following 12 h of drought stress, while genes like MsPIF2 / 3 /5 / 8 / 14 / 15 / 17 were consistently down-regulated. The expression levels of the MsPIF9 /4 /6 in the Zhongmu No. 4 rank within the top three following 12 h of drought stress, as well as six genes (MsPIF1 / 5 /7 / 11 / 12 / 16) also reaching their peak expression at 12 h, while the expression of other genes is down-regulated across all time points. In Xinjiang Daye, the expression levels of MsPIF6 / 9 / 11 / 12 are notably elevated after 12 h of drought stress compared to other time points and other genes.

Fig. 7.

The expression profiles of MsPIF1 to MsPIF8 in four alfalfa cultivars exposed to drought, heat, and combined drought and heat stresses at 0, 6, 12, 24, and 72 h utilizing RT-qPCR. In the representation, the yellow background corresponds to Gannong No. 7 alfalfa, the green background to Zhongmu No. 1, the orange background to Zhongmu No. 4, and the blue background to XinJiang Daye

Fig. 8.

The expression patterns of MsPIF9, MsPIF11 to MsPIF17 in four alfalfa cultivars subjected to drought, heat, and combined drought and heat stresses at 0, 6, 12, 24, and 72 h were analyzed using RT-qPCR. The yellow background represents Gannong No. 7 alfalfa, the green background represents Zhongmu No. 1, the orange background represents Zhongmu No. 4, and the blue background represents XinJiang Daye

In addition to drought stress, we also investigated the response of the PIF family to heat stress (Figs. 7 and 8). In GanNong No. 7, the expression levels of MsPIF4 /6 / 11 are the highest at 12 h of heat stress, with 3/4 MsPIFs genes showing upregulation at both 6 and 12 h of heat treatment before declining. Six genes (MsPIF2 / 3/ 13 / 14 / 15 / 17) in Zhongmu No. 1 showed down-regulated at all 5 time points under stress, while the remaining 10 genes peaked at 12 h post-treatment before being down-regulated. Moreover, the relative expression levels of MsPIF9 / 4 / 6 after 12 h of exposure to heat stress are higher compared to other genes. Similarly, eight genes (MsPIF1 / 4 / 6 / 7 / 9 / 11 / 12 / 16) were up-regulated at 6 and 12 h of stress and then down-regulated at 24 and 72 h in Zhongmu No. 4, while another 8 genes (MsPIF2 / 3 / 5 / 8 /13 /14 / 15 / 17) exhibited decreased relative expression levels at all treatment points. In Xinjiang Daye, nine genes (MsPIF1 / 4 / 6 / 8 / 11 / 13 / 15 / 16 / 17) had their maximum expression at 6 h of heat stress before decreasing, while MsPIF2 consistently showed down-regulation. Additionally, four genes (MsPIF5 / 7 / 12 / 14) had the highest expression at 24 h. Under the combined stress of drought and heat, the relative expression levels of genes MsPIF4 / 6 / 11 in GanNong No. 7 are the highest. Following 12 h of concurrent drought and heat stress, seven genes (MsPIF1 / 2/ 4 / 6 / 9 / 11 / 16) in Zhongmu No. 4 exhibit significantly higher expression levels in contrast to the remaining genes. These results indicate that MsPIFs play an important role in responding to stress resistance in alfalfa, particularly MsPIF4 MsPIF6, and MsPIF9 may be important candidate markers in the response to abiotic stress in the MsPIFs gene family.

Discussion

Numerous evidences have demonstrated that PIFs serve as critical negative regulators of light responses, playing an essential role in various aspects of plant physiology, including light signal transduction, hormone signaling, temperature-induced responses, and responses to abiotic stress pathways [15, 45], it has become a key gene family in unravelling the complexities of plant biology. Understanding the dynamics of this gene family provides a crucial resource for enhancing yields through varietal improvement. The advent of whole-genome sequencing across various plant species has facilitated the delineation of PIFs transcription factors, including those in Arabidopsis (8), pepper (Capsicum annuum) (6), rice (6), soybean (20), maize (7), and wheat (18) [13, 35–37, 46, 47]. Nonetheless, the PIFs transcription factors in alfalfa have not yet been identified and analyzed at the whole-genome scale. This study identified 65 MsPIFs genes and conducted analyses on their structural characteristics, chromosomal localization, phylogeny, gene duplication, and light-responsive cis-elements, along with their expression patterns under various abiotic stresses. The research presents comprehensive insights into the alfalfa PIFs gene family, aiding in the comprehension of functional differentiation among the alfalfa PIFs genes.

In this study, whole-genome scans were carried out on three alfalfa varieties Xinjiang Daye, Zhongmu No. 1, and Zhongmu No. 4, revealing the identification of 29, 9, and 27 MsPIFs genes correspondingly (Table 1). Except for Zhongmu No. 1, the number of MsPIFs genes in the other two alfalfa varieties is significantly higher than that in Arabidopsis and other plants, while similar to soybean. This could be attributed to Xinjiang Daye and Zhongmu No. 4 both being tetraploid, leading to a higher presence of homologous genes in common with soybean. Further analysis found that the grand average of hydropathicity for all 65 MsPIFs proteins is below 0, ranging from − 0.8 to -0.5, indicating their hydrophilic proteins. The PIF protein identified in wheat is also classified as a hydrophilic protein [47]. As transcription factors, PIFs proteins typically operate within the nucleus, where they interact with photoreceptors to modulate plant photomorphogenesis [15]. Subcellular localization analysis identified nuclear localization for 63 out of 65 MsPIFs proteins, with only MsPIF7-X6-1 and MsPIF7-Z17-1 exhibiting different localization patterns (Table 1). In line with subcellular localization findings in related species like wheat [47], potato [48], and maize [49], the subcellular predictions suggest that the majority of MsPIFs genes are situated in the nucleus.

The chromosome mapping analysis reveals an uneven distribution of 65 MsPIFs genes among 5 chromosomes in Xinjiang Daye, 3 chromosomes in Zhongmu No. 1, and 7 chromosomes in Zhongmu No. 4. Notably, chr1 and 7 in Xinjiang Daye exhibit the highest gene counts of 12 and 9, respectively; Chr1 of Zhongmu No. 1 houses 5 genes, while Zhongmu No. 4 chr1 contains 9 genes (Fig. 3). These findings suggest that MsPIFs genes predominantly function on these specific chromosomes. The PIF family genes in Arabidopsis thaliana are primarily located on chromosomes 1, 2, 4, and 5, while those in rice are mainly distributed on chromosomes 1.0 and 3.0. In contrast, the PIF family genes in Medicago truncatula are predominantly found on chromosomes chr1, chr 3, chr 5, and chr 7 (Fig. 5). These results suggest that alfalfa exhibits a more abundant repertoire of PIF homologs compared to the other species studied. In model plants (Arabidopsis thaliana, Oryza sativa, Medicago truncatula), the orthologous genes of the PIF family are typically located on distinct homologous chromosomes in alfalfa. The copy number expansion of these genes in alfalfa may enhance the plant’s adaptive evolution in specific environments by potentially improving light signaling regulation, stress response, or developmental plasticity [50–52]. Gene duplication serves as a primary driving force for the expansion of members within plant gene families, with segmental duplication and tandem duplication representing the two main forms of gene duplication [53]. During the evolutionary process, the alfalfa genome has experienced gene duplication and the accumulation of a large number of transposons, with transposons primarily driving genome expansion, ultimately leading to the enlargement of the alfalfa genome [54]. Previous research has highlighted the pivotal role of segmental duplications in the proliferation of the PIF gene family [55]. This study identified a total of 198 gene replication events across three alfalfa varieties, including 58 sets tandem duplications and 140 pairs segmental duplications (Fig. 4), suggesting a significant influence of fragment repeat events on the MsPIFs gene.

To achieve a more refined classification of the PIF gene family, we performed a comprehensive systematic phylogenetic analysis that incorporated PIF genes from Medicago sativa (Xinjiang Daye, Zhongmu No. 1, Zhongmu No. 4), M. sativa spp. caerulea, Medicago truncatula, Arabidopsis, rice, soybean, and Trifolium repens. Evolutionary tree analysis delineates the division of the PIF gene family into 5 classes and designated as PIF I to PIF V and the PIF II b has the highest MsPIFs members (Fig. 1). The tree showed that the PIF gene family members of alfalfa and soybean (or clover) tend to cluster more closely on the evolutionary tree, while the PIF genes of rice and Arabidopsis thaliana tend to be located more on a different branch from alfalfa. This indicates a closer relationship between alfalfa and soybean (or clover), and a more distant relationship with rice and Arabidopsis thaliana. This could be attributed to the classification of rice within the Poaceae, contrasted with alfalfa, soybean, and clover, which are classified under the Leguminosae, indicating a substantial overlap in their genetic backgrounds. Studies by Wang et al. [56], Chen et al. [57], and Shi et al. [58]. have demonstrated that alfalfa exhibits high homology with Medicago truncatula, while showing weaker phylogenetic relationships with Arabidopsis thaliana and Oryza sativa. Genes from diverse subfamilies display certain differences in the types, numbers, and sequences of conserved motifs and gene structure, while genes within a specific subfamily demonstrate significant level of conservation in their structural characteristics and motifs. For instance, eight genes (MsPIF9-X7-1 / 2 / 3, MsPIF6-Z17-1 / 2, MsPIF7-Z47-1 / 2, MsPIF8-X7-1) within the PIF III b subfamily exhibit consistent arrangements of conservative motifs, each encompassing motif 8, 11, 3, 1, 2, 4, 7, 5, and 12. The structure of these genes is very similar, all consisting of 6 exons and 4 introns, with relatively long introns and gene lengths (Figs. 1 and 2B and C). The structural differences existing between different subfamilies reflect the diversity of the function of Medicago sativa PIFs. The conservation of genes within the subfamily not only reveals common characteristics and similar biological functions of the genes within the subfamily but also provides strong clues for further research on the functional redundancy of genes within the subfamily [59].

The expression levels of target genes are regulated by upstream transcription factors through their binding to cis-acting elements [60]. The alfalfa MYB-like transcription factor (MsMYBH) specifically recognizes the MBS sequence to regulate MsMCP1/MsMCP2, thereby controlling water balance, antioxidant defense, and photosynthesis maintenance under drought conditions, ultimately enhancing the plant’s drought tolerance [61]. The MBS elements in the CER4 and KCS6 genes are key binding sites recognized by PtoMYB142. By activating the expression of these two genes, they significantly promote the biosynthesis of cuticular wax in Populus tomentosa Carr. leaves, thereby establishing a more effective water-retention barrier and ultimately enhancing the plant’s survival capacity in arid environments [62]. In cucumber (Cucumis sativus L.), CsbZIP50 can enhance drought tolerance by specifically binding to the G-box/ABRE cis-acting element in the promoter region of CsRD29 [63]. Further analysis revealed that the promoter region of the alfalfa PIF gene contains many important cis-elements, including components responsive to environmental stimuli such as light, drought, and temperature (Box 4, TC-rich repeats, G-box, MBS, LTR, etc.), along with elements associated with hormone (CGTCA-motif, TGACG-motif, ABRE, etc.) (Fig. 6). The presence of these components in the alfalfa PIFs genes suggest their potential interaction with specific transcription factors, allowing them to effectively sense the dynamic changes in the external environment and hormonal stimuli. Subsequently, these components regulate the expression of the MsPIFs genes, ultimately enhancing the adaptability and stress resilience of alfalfa. The present results align with the established roles of other plant PIFs genes in stress responses [33, 64, 65]. Nevertheless, the size and intricacy of the MsPIFs gene family exceed previous findings in other plant species, potentially introducing a unique stress response mechanism and offering additional targets for enhancing alfalfa’s stress tolerance. It is noteworthy that PIFs genes within the same subfamily harbor similar cis-acting elements, whereas genes across different subfamilies exhibit diverse elements. These results suggest functional diversification among distinct subfamilies, potentially associated with their respective roles in plant development and responses to environmental stresses.

Fig. 6.

The cis-elements of the MsPIFs genes promoter region were examined. The cis-element abundance in each MsPIFs gene promoter region (2 kb upstream of the translation initiation site) was depicted in the grid using distinct colors and numbers

Transcription levels of various MsPIFs under different abiotic stresses were detected using RT-qPCR (Figs. 7 and 8). Studies have demonstrated that AtPIF4 plays a crucial role in the high-temperature signaling pathway. Through its interaction with AtSPCH, AtPIF4 suppresses its expression, thereby limiting stomatal formation under increased temperatures [27, 66]. Phylogenetic tree analysis revealed that MsPIF6 (MsPIF5-Z13-1) exhibits high homology with AtPIF3 (Fig. 1, Table S2). PIF3 plays a crucial role in various biological processes in Arabidopsis thaliana, including light signal transduction, chlorophyll biosynthesis, auxin signal transduction, and cell wall modification [67, 68]. In present study, exposure to high-temperature stress led to substantial alterations in the expression levels of MsPIF4, MsPIF6, MsPIF9, and MsPIF11 after 12 h. These results suggest that MsPIF4, MsPIF6, MsPIF9, and MsPIF11 may play critical roles in alfalfa’s response to abiotic stress.

Numerous studies have demonstrated that PIF family genes can enhance plant tolerance to drought stress by regulating ABA biosynthesis and ABA-responsive genes. The promoter region of ZmPIF1 has been recognized as an abundant reservoir of drought-inducibility (MBS) and abscisic acid responsiveness (ABRE) elements, following drought and abscisic acid treatments, the expression of ZmPIF1 in maize was enhanced [33]. DcPIF3 plays a positive role in drought stress by increasing endogenous ABA levels and promoting the expression of ABA-associated genes [69]. Moreover, MsPIF9 shows high homology with rice OsPIL15 (Fig. 1, Table S2). OsPIL15 has been demonstrated to mediate responses to drought stress in Oryza sativa by regulating ABA-related signaling pathways [70]. In this research, 3 ABRE elements were identified in both Zhongmu No. 4 MsPIF9 (MsPIF5-Z43-1, MsPIF5-Z45-2 / 3) and Xinjiang Daye MsPIF9 (MsPIF6-X5-1 / 2 / 4). By a 12 h period of drought stress, their expression levels increased by 110 and 23-fold, respectively. Furthermore, 2 MBS elements were detected in Zhongmu No. 4 MsPIF9 (MsPIF5-Z45-2), and Xinjiang Daye MsPIF9 (MsPIF6-X5-1). Upon 12 h of drought treatment, the expression levels surged by 110 and 23-fold, respectively. Concurrently, 1 MBS elements were found in Zhongmu No. 1 MsPIF4 (MsPIF3-Z11-1) and MsPIF6 (MsPIF5-Z13-1), Zhongmu No. 4 MsPIF4 (MsPIF4-Z42-1), and Xinjiang Daye MsPIF4 (MsPIF2-X1-1 / 2 / 3 / 4), with the relative expression levels of these four genes increased by 14, 25, 49 and 9-fold compare to 0 h, respectively (Figs. 6, 7 and 8, table S2). Noteworthy is the fact that after 12 h of drought stress, the relative expression levels of MsPIF4 / 6 / 9 genes in Gannong No. 7 rose significantly by 1400, 3500, and 15 times compared to the levels at the onset (0 h) (Figs. 7 and 8). The aforementioned results suggest that MsPIFs play a pivotal role in mediating stress resistance in alfalfa, with particular emphasis on MsPIF4, MsPIF6, and MsPIF9 as potentially crucial candidate genes within the MsPIFs gene family responding to abiotic stress.

Conclusion

A total of 21 MsPIFs genes were identified in the published alfalfa genomes of ‘Xinjiang Daye’, ‘Zhongmu No. 1’, and ‘Zhongmu No. 4.’ These MsPIFs were further categorized into five clades (PIF I to PIF V) and localized into different chromosome. The cis-acting elements of these MsPIF genes are mainly related to plant hormones, stresses (high-temperature, low-temperature, and drought), and light response. The expression levels of MsPIFs significantly altered following exposure to drought and high temperature, particularly evident in the response of the MsPIF4, 6, and 9 genes. Given the critical roles of MsPIF4, MsPIF6, and MsPIF9 in drought and heat stress responses, these genes represent promising candidates for targeted genetic modification strategies to enhance drought and high-temperature tolerance in alfalfa.

Abbreviations

PIFs

Phytochrome-interacting factors

bHLH

Basic helix-loop-helix

RT-qPCR

Real-time quantitative polymerase chain reaction

MDA

Malondialdehyde

EL

Electrolyte leakage

APB

Phytochrome B-binding

APA

Phytochrome A-binding

phyB

Phytochrome B

phyA

Phytochrome A

ABA

Abscisic acid

GA

Gibberellins

BRs

Brassinosteroids

JA

Jasmonic acid

ETH

Ethylene

CBF

C-repeat binding factor

ROS

Reactive oxygen species

chr

Chromosome

Authors’ contributions

HLM and TC conceived and designed the experiment. TC and YW performed the experiments. TC, YW and KJN analyzed all the data. TC wrote the manuscript. HLM revised the manuscript. All authors contributed to the acquisition of data, interpretation of results and critical discussion and approved the final version of the manuscript.

Funding

This work was supported by the Biological Breeding-National Science and Technology Major Project (2022ZD0401102) and the Youth PhD Funding Project From Gansu Provincial Education Department (2024QB-075).

Data availability

All of the datasets supporting the results of this article are included within the article and its Additional files. Nucleotide sequences of Arabidopsis thaliana were acquired from the TAIR website (https://www.arabidopsis.org/), and all nucleotide sequences and accession IDs can be found in the Table S3. The Trifolium repens, Oryza sativa, and Glycine max were obtained from NCBI (https://www.ncbi.nlm.nih.gov/), and all nucleotide sequences and accession IDs can be found in the Table S4. Genomic data for alfalfa varieties (Xinjiang Daye, Zhongmu No. 1, and Zhongmu No. 4), Medicago truncatula, and Medicago sativa spp. Caerulea were sourced from the MODMS website (https://modms.lzu.edu.cn/alfalfa/download/downloadPage).

Declarations

Ethics approval and consent to participate

This article does not contain any studies with animals or humans performed by any of the authors. This study complies with institutional, national and international guidelines and legislation.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.