Genome-wide identification and expression analysis of CBF/DREB1 gene family in Medicago sativa L. and functional verification of MsCBF9 affecting flowering time

Abstract

Background

The C-repeat binding factor (CBF)/dehydration-responsive element binding (DREB1) belongs to a subfamily of the AP2/ERF (APETALA2/ethylene-responsive factor) superfamily, which can regulate many physiological and biochemical processes in plants, such as plant growth and development, hormone signal transduction and response to abiotic stress. Although the CBF/DREB1 family has been identified in many plants, studies of the CBF/DREB1 family in alfalfa are insufficient.

Results

In this study, 25 MsCBF genes were identified in the genome of alfalfa (“Zhongmu No. 4”). These genes were distributed on chromosomes 1, 5, 6 and unassembled scaffolds. Phylogenetics divided the CBF members of Medicago sativa, Arabidopsis thaliana, and Medicago truncatula into six groups, of which group VI had the most MsCBFs members, reaching 52% (13/25). Gene duplication analysis showed that 64% (16/25) of MsCBFs formed tandem duplications, and 32% (8/25) formed segment duplications. The expression pattern of MsCBF9 under different hormone treatments was verified by RT-qPCR, and it was found that MsCBF9 responded to GA3, IAA, SA, and MeJA. Overexpression of MsCBF9 in Arabidopsis significantly delayed the flowering time of Arabidopsis. In contrast, the flowering time of the cbfs mutant was earlier, and overexpression of MsCBF9 also increased the number and size of Arabidopsis rosette leaves.

Conclusion

In this study, the CBF/DREB1 family of alfalfa was comprehensively identified and analyzed, and the function of MsCBF9 in regulating flowering time was studied. This study laid a foundation for further analysis of the function of the CBF family in alfalfa.

Clinical trial number

Not applicable.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12870-025-06081-0.

Background

Transcription factors can regulate the expression of downstream genes by specifically binding to cis-acting elements in the promoter region [1, 2]. Therefore, more research has been done on the role of transcription factors in plants. AP2/ERF (APETALA2/ethylene-responsive factor) transcription factor is one of the most important plant transcription factor families [3, 4]. Among them, DREB (dehydration-responsive element binding) belongs to a subfamily of AP2/ERF superfamily, and contains only one AP2 domain [5–7]. The DREB subfamily can specifically recognize DRE/CRT cis-acting elements or core sequences with DRE elements (CCGAC) and mainly plays a role in cold stress and osmotic stress [8, 9].

Previous studies identified 56 members of the DREB subfamily in Arabidopsis and divided them into A1-A6 groups [10].CBF/DREB1 belongs to the A1 group of the DREB subfamily. This group of members has an iconic sequence with a nuclear localization signal (NLS), PKKRPAGR×KF×ETRHP, upstream of the AP2 domain and a conserved DSAWR motif downstream [11]. CBF/DREB1 has complex functions and can regulate many physiological and biochemical metabolic processes in plants, such as regulating plant growth and development, hormone signal transduction, and responding to various stresses [12–19]. They play an important role in maintaining the normal life cycles of plants.

Plants have a suitable transition time from vegetative to reproductive growth, which can optimize sexual reproduction and productivity [20]. It is well known that hormone signaling can affect plant flowering by regulating the expression of flowering-related genes. For example, salicylic acid (SA) is involved in the regulation of CO, FLC, FT and SOC1 transcription, and SA-deficient arabidopsis shows a late flowering phenotype [21]. In rice, defects in the JA signaling pathway affect floret opening and spikelet development [22]. And GAs can regulate flower development by inhibiting the function of DELLA protein [23].Therefore, it is important to explore the regulation of flowering-related genes and their pathways. In recent years, CBF/DREB1 has been found to regulate flowering time in plants [24–27]. Overexpression of OsDREB1C in rice can cause rice flowers to flower earlier and regulate the expression of flowering-related genes, such as OsFTL2 and OsMADS14 [24]. Overexpressing DgDREB1A in Arabidopsis delayed flowering and showed stronger freezing and drought tolerance [25]. Overexpression of GhDREB1 in Arabidopsis delayed flowering in a GA-dependent manner, and flowering-related genes in different regulatory pathways were also affected by GhDREB1 [26].

Alfalfa (Medicago sativa L.) is an important high-quality forage crop with high economic and ecological value [28]. It has the advantages of high adaptability, high protein content, and rich nutritional value [29, 30]. Alfalfa can be cut several times per year, but with the maturity of alfalfa, its yield increases, and forage quality decreases [31, 32]. Therefore, it is particularly important to select the appropriate cutting time. Suppose the flowering time of alfalfa can be delayed. In that case, the feed quality can be maintained for a longer period of time, and the harvest time can be extended to facilitate management without serious loss of feed quality [33–35]. CBF/DREB1 family members have been identified in a variety of species, including 6, 12, 14 and 10 in Arabidopsis thaliana [10], Lolium perenne [36], Glycine max [37] and Oryza sativa [38], respectively. Although the DREB gene family in alfalfa has been preliminarily analyzed, CBF/DREB1 has not been comprehensively analyzed and the function of family members has not been explored [39]. In order to fill this gap, this study aims to identify and analyze the CBF/DREB1 gene family of alfalfa, and comprehensively describe the characteristics of MsCBF genes through chromosome localization, phylogenetic analysis, conserved domain analysis and cis-acting element analysis. The function of MsCBF9 in regulating plant flowering time was also explored. This study provides a potential reference for understanding the characteristics of the alfalfa CBF/DREB1 family and its regulation of flowering time.

Results

Identification of CBF genes in the alfalfa genome

Twenty-five MsCBFs were identified in the alfalfa genome (M. sativa L. “Zhongmu No. 4”) using BLASTP, and the protein sequences with two characteristic amino acid sequences, PKK/RPAGR×KF×ETRHP and DSAWR, before and after the AP2 conserved domain, with the 14th position as V (valine) and the 19th position as E (glutamic). These members were named MsCBF1–MsCBF25, based on their chromosomal locations (Fig. 1). Chromosome localization analysis showed that 25 MsCBF genes were unevenly distributed on Chr1, Chr5, Chr6, and unassembled scaffold chromosomes, and 72% of the MsCBF genes were clustered on Chr6.

Fig. 1.

Chromosome distribution of the alfalfa CBF gene. MsCBFs were mapped on chromosomes 1, 5 and 6 of alfalfa. These genes were renamed as MsCBF1 ~ MsCBF25 according to the order of MsCBFs on chromosomes. The vertical bar represents the chromosome of alfalfa, and the scale represents the chromosome length (Mb). Color gradient represents gene density, and red to blue represents high density to low density. The red semicircle represents tandem replication

The gene ID, full CDS length, protein length, molecular weight (MW), isoelectric point (pI), and subcellular location of the 25 MsCBF genes are shown in Table 1 and S1. The results showed that the length of MsCBFs varied greatly, ranging from 396 bp (MsCBF2) to 1233 bp (MsCBF22), with 76% (19/25) of the sequences being between 500 bp and 900 bp. The protein sequences of MsCBF transcription factor family members ranged in length from 131 aa (MsCBF2) to 410 aa (MsCBF22), with a mean length of 244 aa. The molecular weight (MW) of the MsCBF proteins ranged from 14.74 kDa (MsCBF2) and 46.75 kDa (MsCBF22). The isoelectric points ranged from 5.1 (MsCBF12) to 9.86 (MsCBF2), with 60% (15/25) of the proteins were acidic (pI < 7.0) and 40% (10/25) were alkaline (pI > 7.0), indicating that MsCBF proteins were mostly acidic. According to the Subcellular localization prediction website, MsCBF proteins were located in the nucleus or cytoplasm, and 76% (19/25) of the proteins were distributed in both the cytoplasm and the nucleus.

Table 1.

Characteristics of MsCBF genes in Medicago sativa

Gene Name	Gene ID	Full CDS length (bp)		Protein		Subcellular Location
			Length (aa)	Mw (kDa)	pI
MsCBF1	Msa0049840	672	223	25.25	7.59	Cytoplasm. Nucleus.
MsCBF2	Msa0753830	396	131	14.74	9.86	Nucleus.
MsCBF3	Msa0795170	645	214	24.15	8.53	Cytoplasm. Nucleus.
MsCBF4	Msa0795580	645	214	24.15	8.53	Cytoplasm. Nucleus.
MsCBF5	Msa0891960	693	230	26.10	5.35	Cytoplasm. Nucleus.
MsCBF6	Msa0891970	681	226	25.74	5.59	Cytoplasm. Nucleus.
MsCBF7	Msa0891980	687	228	25.85	5.55	Cytoplasm. Nucleus.
MsCBF8	Msa0891990	777	258	29.41	7.67	Cytoplasm. Nucleus.
MsCBF9	Msa0892020	591	196	22.42	5.28	Cytoplasm. Nucleus.
MsCBF10	Msa0892030	843	280	32.11	9.2	Cytoplasm. Nucleus.
MsCBF11	Msa0893350	657	218	24.84	5.29	Cytoplasm. Nucleus.
MsCBF12	Msa0893360	603	200	22.92	5.1	Cytoplasm. Nucleus.
MsCBF13	Msa0928860	816	271	30.77	7.13	Cytoplasm.
MsCBF14	Msa0928870	912	303	34.88	7.97	Cytoplasm.
MsCBF15	Msa0928880	804	267	30.48	6.67	Nucleus.
MsCBF16	Msa0928940	492	163	18.69	6.76	Cytoplasm. Nucleus.
MsCBF17	Msa0956890	792	263	29.80	7.12	Cytoplasm. Nucleus.
MsCBF18	Msa0956900	648	215	24.56	6.66	Cytoplasm. Nucleus.
MsCBF19	Msa0992060	909	302	33.83	5.95	Cytoplasm. Nucleus.
MsCBF20	Msa0992110	678	225	25.63	6.54	Cytoplasm. Nucleus.
MsCBF21	Msa0992130	909	302	33.83	5.95	Cytoplasm. Nucleus.
MsCBF22	Msa0992230	1233	410	46.75	6.18	Cytoplasm. Nucleus.
MsCBF23	Msa1344910	876	291	33.31	6.33	Cytoplasm.
MsCBF24	Msa1344920	816	271	30.73	7.1	Cytoplasm.
MsCBF25	Msa1413960	618	205	23.27	5.41	Cytoplasm. Nucleus.

CDS: coding sequence; bp: base pair; aa: amino acid; MW: molecular weight; pI: isoelectric point

Phylogenetic relationships analysis of MsCBFs

To understand the classification and evolutionary relationships of MsCBF in alfalfa, this study constructed phylogenetic trees of M. sativa and two model plants (A. thaliana and M. truncatula) using the MEGA software (Fig. 2). The results showed that 47 CBF proteins in M. sativa (25 proteins), A. thaliana (6 proteins), and M. truncatula (16 proteins) could be divided into six groups (Groups I–VI). The CBF members of M. sativa were clustered in groups I, III, V, and VI, of which 52% (13/25) were MsCBF members. Only two proteins of A. thaliana (AtDREB1E and AtDREB1F) can be grouped together with M. sativa and M. truncatula, and the other four proteins can be grouped together separately. In M. truncatula, except MtCBF7 and MtCBF14, MtCBF proteins can be grouped together with the CBF proteins of M. sativa. These results suggest that the CBF members of M. sativa and M. truncatula are evolutionarily closer than those of A. thaliana. Then, 25 MsCBFs were divided into four groups according to the phylogenetic tree, and their gene structure and motif composition were analyzed (Fig. 3A).

Fig. 2.

Phylogenetic tree of CBF in M. sativa, A. thaliana, and M. truncatula. The phylogenetic tree was constructed by neighbor-joining of MEGA 6.0. The phylogenetic tree was divided into six ancestors: groups I-VI. Different markers were used to distinguish each species, with blue circles, red triangles, and green stars representing A. thaliana, M. truncatula, and M. sativa, respectively

Fig. 3.

Phylogenetic relationship, motif composition, and gene structure analysis of the MsCBF family. (A) The phylogenetic tree of alfalfa MsCBFs was constructed using the neighbor-joining method in MEGA 6.0. Different colors represent different groups. (B) The gene structure of MsCBFs. The gray box, black box, and black line represent the UTR, CDS, and intron, respectively. (C) AP2 conserved domain of MsCBFs. (D) The MEME program was used to analyze the motif composition of MsCBFs. Different colors were used for each domain. (E) Sequence logos of Motif 1-Motif 10

Gene structure and conserved motif analysis

The gene structure analysis of 25 MsCBF genes in alfalfa was performed by TBtools (Fig. 3B). The results showed that the number of exons in MsCBFs ranged from 1 to 5, the number of introns ranged from 0 to 4, and 72% (18/25) of the sequences had no more than two exons. None of the MsCBFs in Groups I and II had introns in their gene structure.

In order to further understand the conservation and diversity of the MsCBFs transcription factor family in evolution, NCBI-CDD search revealed that MsCBFs proteins all have only one AP2 domain (Fig. 3C). Analysis of conserved motifs (Fig. 3D) showed that members of the MsCBF transcription factor family had similar conserved motifs and arrangements. All MsCBF proteins contained Motif 1 and Motif 2, which were two conserved amino acid sequences of the AP2/ERF domain, respectively (Fig. 3E). The details of the ten motifs are provided in Table S2. Motif 1, a YRG element is the core of the AP2 domain and is involved in the specific binding of DNA. Motif 2, the RAHD element is responsible for regulating the binding strength.

Analysis of the gene duplication and synteny of the MsCBFs

To better understand potential gene duplication events, TBtools was used to visualize tandem and segmental duplication events of MsCBF genes. The results showed that 64% (16/25) of the MsCBFs formed five tandem duplication events, including two tandem duplication events containing four genes (MsCBF5, MsCBF6, MsCBF7 and MsCBF8) (MsCBF13, MsCBF14, MsCBF15, and MsCBF16) and four tandem duplication events containing two genes (MsCBF9 and MsCBF10, MsCBF11 and MsCBF12, MsCBF17 and MsCBF18, MsCBF23, and MsCBF24) (Fig. 1). The collinearity analysis of the alfalfa genome showed that 32% (8/25) of the MsCBF genes formed four segmental duplication events on Chr6 (Fig. 4). The four pairs of segment duplication events are listed in Table S3. These results indicate that the reasons for the expansion of the MsCBF gene family may include tandem and segmental duplication events.

Fig. 4.

The syntenic relationship diagram of MsCBF genes in alfalfa. The blue circle represents the alfalfa chromosome, and the position of the MsCBF gene is shown on the circle. The gray band represents the syntenic regions in the alfalfa genome, and the red band represents the segment replication event

Next, collinearity maps of alfalfa with A. thaliana and M. truncatula were constructed (Fig. 5). Two MsCBF genes (MsCBF2 and MsCBF5) were found to be involved in the formation of four homologous gene pairs in the collinearity analysis of alfalfa and A. thaliana, of which three pairs contained the MsCBF2 gene. In the collinearity analysis of alfalfa and M. truncatula, nine MsCBF genes were found to be involved in the formation of eleven pairs of homologous gene pairs. The number of collinear genes between alfalfa and M. truncatula was approximately three times higher than that in A. thaliana. Gene pairs are shown in Tables S4 and S5. The above results show that the CBF genes of these three plants differentiated during the long-term evolution of the species, but compared with A. thaliana, the number of homologous pairs between alfalfa and M. truncatula was higher, and the MsCBF genes involved were wider. This may be due to the closer genetic relationship between alfalfa and M. truncatula, and the higher homology between MsCBF and MtCBF genes.

Fig. 5.

Collinearity analysis of the MsCBF genes in A. thaliana and M. truncatula. The orange and green boxes represent the chromosomes of alfalfa and model plants, respectively. The gray line represents the syntenic block between alfalfa and the model plants, and the red line represents segmental duplication events of CBF gene pairs

Prediction of cis-acting elements in MsCBFs promoters

To further clarify the biological function of the MsCBF genes in alfalfa, cis-acting elements were predicted in the promoter region (2 kb upstream of the coding region) of MsCBF family members using the PlantCARE database (Fig. 6). According to the prediction results, four types of elements were identified: light, growth and development, hormone, defense and stress. In detail, 84% of MsCBF genes contain AAGAA-motif elements, and 64% contain as-1 elements, which are related to growth and development. In defense and stress elements, all MsCBF members contained MYB, MYC elements, 96% members contained ARE elements, and 76% members contained STRE and WRE3 elements. Additionally, different MsCBFs contain different hormone response elements, indicating that the expression of these MsCBF genes may be induced by different hormones. These results suggest that MsCBF genes may respond to a variety of stress conditions and may also be involved in photoperiod- and hormone-induced growth, development, and flowering regulation.

Fig. 6.

Prediction of cis-acting elements of CBF genes promoter in alfalfa. Cis-acting elements are divided into light, growth and development, hormones, defense, and stress-related elements. The numbers in the boxes represent the number of elements. The color gradient on the right-hand side represents the number of elements. The darker the color of the box, the greater the number of elements

Expression analysis of the MsCBF9 gene under different hormone treatments

Plant hormones play important roles in the regulation of plant growth and development. Therefore, we used different hormone treatments to explore the expression patterns of MsCBF9 (Fig. 7). After treatment with 200 µM GA3, the expression of MsCBF9 was significantly decreased, and the expression level at 72 h was decreased by 53 times compared with the control. After treatment with 200 µM MeJA, the expression of MsCBF9 showed a downward trend as a whole, decreasing by 160 times at 72 h. After treatment with 200 µM IAA, the expression of MsCBF9 increased first and then decreased. The expression of MsCBF9 was significantly activated from 5 to 24 h and decreased after 48 h. After treatment with 200 µM SA, the expression of MsCBF9 decreased significantly at 3 h, began to recover at 5 h, and decreased again at 24 h.

Fig. 7.

The expression profiles of MsCBF9 under 200µM GA3, MeJA, IAA and SA treatments. The horizontal coordinates represent the processing time (0 h, 3 h, 5 h, 8 h, 12 h, 24 h, 48 h, 72 h), and the vertical coordinates represent relative expression levels. The error bar represents the standard error of the mean of three independent repetitions. ANOVA with p < 0.05. There were significant differences between different letters (a-i)

Flowering phenotype identification in transgenic Arabidopsis

In order to study the function of the MsCBF9 gene in regulating flowering, MsCBF9 was heterologously expressed in A. thaliana (Fig. 8). It was found that under long-day conditions, the flowering time of transgenic lines OE-1 and OE-20 was 1–3 days later than that of WT, and the flowering time of the cbfs mutant was two days earlier than that of WT (Fig. 8A, D). On the 30th day of growth, the plant height of the two overexpression lines was significantly lower than that of WT, the rosette leaves of OE-1 and OE-20 were larger, and the number of leaves was also five and three more than that of WT, respectively (Fig. 8B, C, E, F). The plant height of the cbfs mutant was significantly higher than that of the WT, but there was no significant difference in the number of leaf leaves between the cbfs mutant and WT plants. Through qRT-PCR detection of flower-promoting related genes (FT, LFY, SOC1, FCA) of A. thaliana in transgenic lines, WT and cbfs mutant, it was found that these genes were significantly inhibited in transgenic lines, while significantly up-regulated in cbfs mutants (except LFY was not significantly different from WT) (Fig. 9).

Fig. 8.

Regulation of flowering period by overexpression of MsCBF9 in Arabidopsis under long-day conditions. (A) The status of WT, overexpressing MsCBF9 lines OE-1, OE-20 and cbfs mutants at 30 days of growth. (B) (C) The size and morphology of Arabidopsis rosette leaves of WT, overexpressing MsCBF9 lines OE-1, OE-20, and cbfs mutants at 30 days of growth. (D) WT, overexpressing MsCBF9 lines OE-1, OE-20 and cbfs mutant flowering time statistics. Each line had 9 biological replicates. (E) Plant height statistics of WT, overexpressing MsCBF9 lines OE-1, OE-20, and cbfs mutants at 30 d of growth. Each line had 9 biological replicates. (F) The number of rosette leaves of WT, overexpressing MsCBF9 lines OE-1, OE-20, and cbfs mutants was counted after 30 days of growth. Each line had 9 biological replicates. The asterisk in the column indicates a significant difference between the WT and transgenic lines (*p < 0.05; **p < 0.01, t-test)

Fig. 9.

Expression levels of flowering-related genes (FT, LFY, SOC1, and FCA) in Arabidopsis thaliana. The horizontal coordinates represent different genes, and the vertical coordinates represent the relative expression levels. The error bar represents the standard error of the mean of three independent repetitions. The asterisk in the column indicates a significant difference between the WT and transgenic lines (*p < 0.05; **p < 0.01, t-test)

Discussion

CBF/DREB1 transcription factors exist only in plants and play an important role in regulating plant growth and development and responding to abiotic stresses [8, 18, 40]. CBF/DREB1 family members have been identified in many species, including 6, 12, 14, 6, and 10 in A. thaliana [10], Lolium perenne [36], Glycine max [37], Camellia sinensis [41] and Taraxacum kok-saghyz [42]. In our study, 25 MsCBF genes were identified in the genome of alfalfa “Zhongmu No.4”, and these genes were comprehensively analyzed for protein characteristics, phylogenetic relationships, gene duplication, gene structure, conserved domains and cis-acting elements. The regulation of flowering time by MsCBF9 was explored.

In this study, a phylogenetic tree of CBF proteins in M. sativa, M. truncatula, and A. thaliana was constructed, which could be divided into six groups: Group I-Group VI. The alfalfa CBF members were clustered in groups I, III, V, and VI. MsCBF1-MsCBF4 was clustered with AtDREB1E and AtDREB1F. Previous studies have shown that AtDREB1E and AtDREB1F in Arabidopsis are related to salt and drought stress [43], so it is speculated that MsCBF1-MsCBF4 may also be related to salt and drought stress. Chromosomal localization analysis showed that 11 MsCBFs were clustered on chromosome 6 of alfalfa. Previous studies also found that MtCBFs were clustered on chromosome 6 of M. truncatula, indicating that these genes may synergistically regulate downstream genes or functional redundancy [44]. Genome-wide duplications play an important role in the amplification of members of many gene families. Tandem duplication and segmental duplication are the two main forms of duplication [45]. In this study, we found that 64% of MsCBFs formed tandem duplication events, whereas only 32% formed segmental duplication events, indicating that tandem duplication may be the main driving force for expanding the CBF/DREB1 gene family. The phenomenon in which CBFs genes are arranged in tandem on chromosomes has also been reported previously. For example, CBF genes in Triticeae genomes are arranged in tandem on chromosome 5, and CBF genes in Zea mays genomes are arranged in tandem on chromosome 7 [46, 47]. Even a relatively small number of Oryza sativa CBF genes show a tandem arrangement on chromosome 9 [48]. Analysis of the conserved domains of MsCBFs revealed that Motif1 and Motif2 contain the characteristic conserved elements YRG and RAHD of the AP2 domain, respectively. These two elements play important roles in the binding of CBF transcription factors to specific downstream DNA sequences [49].

Plant hormones play an important role in regulating plant growth and development, and previous studies have shown that they contribute to the regulation of plant flowering. The GA pathway is one of six plant flowering pathways [50, 51]. The exogenous addition of GA can accelerate the flowering of Arabidopsis under short-day conditions, and it was found that the gai-1 mutant flowered extremely late under short-day conditions [52, 53]. Exogenous IAA can induce flower stalk elongation in Brassia campestris [54]. MeJA treatment can promote the opening of sorghum florets by regulating genes related to metabolic pathways and plant hormone signal transduction pathways in sorghum floret plasma cells [55]. Studies have also shown that SA is associated with many flower developmental processes, such as pollen germination and pollen tube elongation [56, 57]. This study also explored whether MsCBFs responded to various plant hormones. The results showed that MsCBF9 responded to IAA, GA, SA and MeJA.Under the treatment of 200 µM IAA, GA3, SA and MeJA, the expression of MsCBF9 was significantly inhibited. These results suggest that MsCBF9 may regulate plant flowering by influencing the hormone signaling process. Next, this study heterologously expressed MsCBF9 in Arabidopsis to further explore the regulation of this gene during flowering. Overexpression of MsCBF9 in Arabidopsis significantly delayed the flowering time of Arabidopsis, while the flowering time of cbfs mutants was significantly advanced. Overexpression of MsCBF9 also increased the number of rosette leaves in Arabidopsis. Previous studies have shown that CBF genes can delay flowering in plants. For example, heterologous expression of LcCBF2 and LcCBF3 in Arabidopsis can lead to delayed flowering [58]. It has been reported that the CBF gene causes flowering delay, which may be partially attributed to its activation of FLC and subsequent inhibition of the two flowering pathway integrators FT and SOC1 [59]. This finding is consistent with the results of the present study. The expression of FT and SOC1 genes in the two overexpression lines was significantly inhibited, while the expression of these two genes in the cbfs mutant was significantly increased. Other flowering-related genes, such as LFY and FCA, have the same expression pattern.

Conclusions

A total of 25 MsCBF genes were identified in this study, which were mainly distributed on chromosomes 1, 5 and 6. Among them, 64% of the genes formed tandem repeats, indicating that tandem duplication may be the main driving force for MsCBFs amplification. Subcellular localization prediction showed that MsCBF protein was located in the nucleus and cytoplasm. Further analysis of the domains shows that MsCBF members located in the same branch of the evolutionary tree contain similar domains. Moreover, each MsCBF contains YRG and RAHD elements to ensure that the CBF protein can function normally. Through the analysis of MsCBFs promoter, it was found that MsCBFs contained many cis-acting elements related to hormones, growth and development. MsCBF9 was selected for subsequent research, and it was found that the gene responded to four hormones: IAA, GA, SA and MeJA. Heterologous expression of MsCBF9 in Arabidopsis can significantly delay the flowering time of Arabidopsis, while the flowering time of cbfs mutant plants is significantly earlier. This study provides preliminary insights into the molecular basis of the CBF gene family in alfalfa. However, the mechanism by which MsCBF9 regulates flowering and the functions of other MsCBF members are still unclear. Therefore, future research will focus on the analysis of the function and specific regulatory mechanisms of MsCBFs.

Methods and materials

Identification of CBF Family members in alfalfa

The CBF/DREB1 protein sequences of A. thaliana were obtained from TAIR website (https://www.arabidopsis.org/, accessed on 8 January 2024). The genome and annotation information of alfalfa (Cultivar “Zhongmu No.4”) was downloaded from the figshare data repository (https://figshare.com/s/fb4ba8e0b871007a9e6c, accessed on 8 January 2024). The CBF/DREB1 protein sequences of M. truncatula were obtained from EnsemblPlants website (https://plants.ensembl.org/index.html, accessed on 8 January 2024). AtCBF1 (AT4G25490), AtCBF2 (AT4G25470), AtCBF3 (AT4G25480), AtCBF4 (AT5G51990), AtDREB1E (AT1G63030), AtDREB1F (AT1G12610) protein sequences were used as query sequences to perform BLASTP in TBtools software with 1 × 10^− 10 cutoff E-values against the alfalfa reference genome. Then, NCBI-CDD (https://www.ncbi.nlm.nih.gov/cdd, accessed on 21 January 2024) was used to search for protein sequences containing only one AP2 domain. Clustal Omega (https://www.ebi.ac.uk/jdispatcher/msa/clustalo, accessed on 21 January 2024) of EBI (European Bioinformatics Institute) was used for multiple sequence alignment to find two characteristic amino acid sequences PKK/RPAGR×KF×ETRHP and DSAWR before and after the AP2 conserved domain, and the 14th V (valine) and 19th E (glutamic acid) in the AP2 domain. The protein sequences with these characteristics belong to the CBF/DREB1 gene family. Expasy (https://www.expasy.org/about) was used to remove redundancy of the obtained MsCBF protein sequence, with default values for all parameters. Finally, 25 CBF family members were identified in alfalfa. The predicted isoelectric (pI) and molecular weights (MWs) of all MsCBF genes were predicted with the Expasy-ProtParam website (https://web.expasy.org/protparam/, accessed on 29 January 2024). Plant-mPLo (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/, accessed on 29 January 2024) was used to predict subcellular localization.

Chromosome location analysis and phylogenetic tree construction

The genomic location of the identified MsCBF was confirmed and mapped using Gene Location Visualize from the GTG/GFF of TBtools software (v1.108, Chen, C., GZ, China) [60]. Using the ClustalW program in MEGA 6.0 software (Tamura, K., Tokyo, Japan) [61], full-length amino acid sequences were aligned, and a phylogenetic tree was constructed with CBF protein sequences from alfalfa, M. truncatula, and A. thaliana using the neighbor-joining (NJ) method (bootstrap values for 1000 replicates, other parameters are selected by default). The iTOL website was used to beautify phylogenetic trees (https://itol.embl.de/, accessed on 3 February 2024).

Gene structure and conserved domains analysis

The MEME online program (https://meme-suite.org/meme/doc/meme.html, accessed on 8 February 2024) was used to analyze conserved motifs in MsCBF proteins, and the maximum number of motifs was 10 [62]. The structural (intron-exon) information of MsCBFs were included in the alfalfa GFF file. Both conserved motifs and gene structures were visualized using TBtools (v1.108, Chen, C., GZ, China).

Cis-acting elements analysis of MsCBFs promoter

The 2 kb upstream of the coding region of MsCBF genes was obtained from the alfalfa genome database. Extracted sequences were submitted to the PlantCARE website (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 24 February 2024) for cis-acting element analysis, and visualized using TBtools (v1.108, Chen, C., GZ, China) [63].

Gene duplication and synteny analysis

One-step MCScanX-Super fast of TBtools (v1.108, Chen, C., GZ, China) with default parameters was used to analyze the pattern of gene duplication of the MsCBFs. The Dual Synteny Plot was used to determine the syntenic relationship between orthologous MsCBFs and other species.

Plant materials and growth conditions

The alfalfa seeds “Zhongmu No. 4” were obtained from the Institute of Animal Science of the Chinese Academy of Agricultural Sciences. The growth conditions were consistent with those previously reported [29]. After three weeks, alfalfa was treated with hormones. The experimental groups were treated with 1/2 Hoagland solution supplemented with 200 µM GA3, SA, IAA, and MeJA. The control group only added 1/2 Hoagland solution. The leaves of the seedlings were collected at 0, 3, 5, 8, 12, 24, 48, and 72 h and stored at − 80 °C.

Cloning and expression vector construction of MsCBF9

Total RNA was extracted from alfalfa leaves using a Plant Total RNA Extraction Kit (Promega, Madison, WI, USA), and cDNA was obtained by reverse transcription. According to the genome database of alfalfa “Zhongmu No.4”, the full-length CDS sequence of MsCBF9 (Gene ID: Msa0892020) was extracted for gene synthesis, and the pUC57-MsCBF9 plasmid was obtained. The specific primer Super-MsCBF9-F/R, containing the homologous arm, was designed by selecting the SmaI restriction site, and the target sequence was amplified from the pUC57-MsCBF9 plasmid. Primer sequences are shown in Table S7. The amplified product was recovered and seamlessly cloned into an expression vector to construct a Super-MsCBF9-GFP expression vector to obtain a recombinant plasmid. The correctly sequenced plasmid was transformed into Agrobacterium GV3101 (Huayueyang Biotechnology, Beijing, China) and heterologously transformed into Arabidopsis. Transgenic Arabidopsis was screened on 1/2MS medium containing 4 mg/ml phosphinothricin (PPT) (Coolaber, Beijing, China), and PCR detection was performed. Positive plants were selected for screening until the homozygous plants were obtained.

Flowering phenotype identification in transgenic Arabidopsis

WT, transgenic lines OE-1, OE-20, and cbfs mutant plants were planted on 1/2MS medium, placed at 4 °C for three days of vernalization, and moved to the incubator after three days. After ten days, it was moved into the soil. Nine seedlings were planted per pot. The first day of growth was recorded when seeds germinated on 1/2MS medium. At 30 days, the flowering status of the different lines was observed and photographed, and the plant height and number of rosette leaves were counted.

Real-time quantitative PCR (RT-qPCR) analysis

The RT-qPCR method was consistent with a previous study [64]. Primer sequences are shown in Table S6. Three technical replicates were performed for each biological repetition. RT-qPCR was used to analyze the relative gene expression using the 2^−ΔΔCt method.

Statistical analysis

SPSS software was used for statistical analysis. Duncan multiple range test was used to analyze differences between groups (ANOVA with p < 0.05). A t-test was used to determine whether there was a significant difference between the two samples (*p < 0.05; **p < 0.01). GraphPad Prism 9 (GraphPad Software, Boston, FL, USA) and Adobe Illustrator CC 2020 (ADOBE, San Jose, CA, USA) were used for the drawing and graphic editing, respectively.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Abbreviations

CBF

C-repeat binding facto

DREB

Dehydration-responsive element binding

DREB1

Dehydration-responsive element binding 1

AP2

APETALA2

ERF

Ethylene-responsive factor

CDS

Coding sequence

bp

Base pair

aa

Amino acid

MW

Molecular weight

pI

Isoelectric point

Mb

Chromosome lengt

RT

qPCR Real-Time Quantitative PCR

Author contributions

Experimental design and planning and first draft writing, J.C., Y.L., J.K.; preparation and modification of the images, H.L.; data processing, manuscript modification, X.J., L.Z.; data analysis and test data accuracy, H.D., X.W.; application and analysis of the software used in the experiment, F.H., M.L.; data and manuscript review, J.K.; funding acquisition, J.K. All the authors contributed to the article. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (32071868,32371770) and the National Key Research and Development Program of China (2022YFF1003203).

Data availability

All data generated or analyzed during this study are included in this published article and its supplementary information files.

Declarations

Ethics approval and consent to participate

Field and laboratory studies were conducted through local legislation. This article does not include any research involving human participants or animals, nor any endangered or protected species. The plant materials collected and the experiments conducted in this study are consistent with institutional, national and international guidelines and legislation.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.