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Genome-wide identification of SHMT family genes in alfalfa (Medicago sativa) and its functional analyses under various abiotic stresses

BMC Genomics volume 25, Article number: 781 (2024) Cite this article

Abstract

Background

Alfalfa (Medicago sativa L.) is the most widely planted legume forage and one of the most economically valuable crops in the world. Serine hydroxymethyltransferase (SHMT), a pyridoxal phosphate-dependent enzyme, plays crucial roles in plant growth, development, and stress responses. To date, there has been no comprehensive bioinformatics investigation conducted on the SHMT genes in M. sativa.

Results

Here, we systematically analyzed the phylogenetic relationship, expansion pattern, gene structure, cis-acting elements, and expression profile of the MsSHMT family genes. The result showed that a total of 15 SHMT members were identified from the M. sativa genome database. Phylogenetic analysis demonstrated that the MsSHMTs can be divided into 4 subgroups and conserved with other plant homologues. Gene structure analysis found that the exons of MsSHMTs ranges from 3 to 15. Analysis of cis-acting elements found that each of the MsSHMT genes contained different kinds of hormones and stress-related cis-acting elements in their promoter regions. Expression and function analysis revealed that MsSHMTs expressed in all plant tissues. qRT-PCR analysis showed that MsSHMTs induced by ABA, Salt, and drought stresses.

Conclusions

These results provided definite evidence that MsSHMTs might involve in growth, development and adversity responses in M. sativa, which laid a foundation for future functional studies of MsSHMTs.

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Introduction

Alfalfa (Medicago sativa L.), originally from southwestern Asia, is a perennial legume widely grown around the world as an economic forage, and is known as the “King of Forages”. Alfalfa is rich in protein, vitamins, flavonoids and other nutrients [1]. In addition, alfalfa has a well-developed root system, and its rhizobacteria can not only provide nitrogen nutrition for plants, but also increase soil fertility and improve soil structure, so it has become the preferred pasture for ecological projects such as returning farmland to grassland, management of wind and sand sources, and soil and water conservation. In recent years, with the change of global climate and soil, the development of alfalfa industry is seriously limited by adverse environmental conditions [2]. During the seedling stage, salinity stress resulted in a significant decrease in root length, root surface area, and root volume of alfalfa [3]. In addition, high salt concentrations resulted in water loss from leaf cells, reduced primary cell elongation, decreased growth rate, and significantly reduced leaf area [4]. Maroua et al. found that under abiotic stress, alfalfa not only has a significant decrease in fresh weight and dry weight yield, but also a significant decrease in protein content [5].

Serine hydroxymethyltransferase (SHMT, EC 2.1.2.1), a pyridoxal phosphate-dependent enzyme, catalyzes the interconversion and reversible hydroxymethyl transfer between L-serine/glycine and tetrahydrofolate (H4PteGlun, THF)/5,10-methylenetetrahydrofolate (5,10-CH2-H4PteGlun). To date, SHMTs have been reported in various plants, including A. thaliana [6], S. lycopersicum [7], G. max [8], O. sativa [9], T. aestivum [10], M. truncatula [11], C. sativus [12], and P. trichocarpa [13]. Research has revealed that SHMTs are distributed across multiple cellular compartments, including mitochondria, cytoplasm, chloroplast, and the nucleus [14]. Mitochondrial-localized SHMT has been extensively studied, particularly in A. thaliana, where SHMT1 regulates sucrose accumulation and reactive oxygen species (ROS) homeostasis, crucial for primary root growth [15]. Reducing the protein activity of SHMT1 by controlling the level of phosphorylation at S31 leads to decreased metabolic levels, proline and polyamine accumulation, and stomatal closure, which reduces salt tolerance in A. thaliana [6]. Heterologous overexpression of AtSHMT1 promotes formaldehyde metabolism in tobacco leaves. Additionally, co-overexpression of AtSHMT1 and AtFDH induces sugar synthesis and enhances the role of original pathways during formaldehyde metabolism in tobacco [16]. In most cases, mitochondrial SHMT engages in the formation of multiprotein complexes, thereby participating in plant growth, development, and stress response processes. For instance, the mitochondrial SlSHMT interacts with chaperonin 60α1 (SlCPN60α1) to regulate the process of photosynthesis and photorespiration in S. lycopersicumes [17]. GmSHMT08 forms a multiprotein complex with soluble NSF attachment protein GmSNAP18 and pathogenesis-related protein GmPR08-Bet VI, maintaining cellular redox homeostasis and conferring resistance to G. max cyst nematode (SCN) [18]. OsSHMT1 forms a multiprotein complex with NLR protein OsRSR1, providing resistance against O. sativa sheath blight disease [19]. Research on the functionality of SHMT in other cellular has been relatively limited. The OsSHMT located in the endoplasmic reticulum (ER) enhances cold tolerance in O. sativa by interacting with APX and heat shock protein 70 (Hsp70), facilitating the removal of excess H2O2 in plants [20]. Moreover, transient silencing of TaSHMT3A-1 reduces T. aestivum resistance to fusarium head blight [10]. A study on PtSHMT2 localized in the cytoplasm showed its high expression levels within the wood tissue during P. trichocarpa development and demonstrated that overexpression PtSHMT2 enhances P. trichocarpa growth through increased biomass production and the release of sugars (glucose and xylose) [13].

No SHMT family member in M. sativa has been characterized till date. Thus, in order to discover the genetic evolution and function of SHMT gene family in M. sativa, we utilized the published genomic information of M. sativa (cultivar XinJiangDaYe) to identify members of the MsSHMT genes. The expansion patterns, evolutionary relationships, gene structures, conserved motifs, cis-acting elements, as well as tissue-specific expression patterns of the MsSHMTs were systematically analyzed. Additionally, the response patterns of the MsSHMT genes to harmonizes and stresses were analyzed using qRT-PCR. These findings establish a theoretical foundation for understanding the functionality of MsSHMTs and provide important candidate genes for resistance studies in M. sativa.

Materials and methods

Identification of the SHMT gene family in M. sativa

First, we obtained the Hidden Markov Model (HMM) of the SHMT domain (PF00464) from the Pfam database (https://pfam.xfam.org/, accessed on 22 August 2023). Meanwhile, the genome assembly files of M. sativa (cultivar XinJiangDaYe, Released in December 2020) were downloaded from the figshare projects (https://figshare.com/projects/whole_genome_sequencing_and_assembly_of_Medicago_sativa/66380, accessed on 22 August 2023) [21]. Then, taking the HMM of SHMT as a template, we utilized HMMER to query the candidate proteins from the M. sativa database with an e-value cut-off of e− 10. The redundant sequences of candidate proteins were removed using the CD-Hit with threshold 90% [22]. The physical and chemical properties were analyzed by Expasy (https://web.expasy.org/protparam/, accessed on 28 August 2023) [23]. Subcellular localization was predicted using the online website WOLF-PSORT (https://wolfpsort.hgc.jp/, accessed on 28 August 2023).

Exon-Intron structure analysis and conserved motifs identification

The exon-intron structure information of MsSHMTs was extracted from the M. sativa general feature format (GFF) file. The conserved motif of MsSHMTs was identified by the online tool MEME (https://meme-suite.org/meme/, accessed on 10 September 2023), with default setting except for changes in the number of motifs to 10 [24]. Results were visualized using TBtools.

Phylogenetic analysis

A total of 76 SHMT protein sequences from A. thaliana (7), G. max (13), O. sativa (5), P. trichocarpa (9), S. lycopersicum (7), C. sativus (6), T. aestivum (14), and M. sativa (15) were downloaded from TAIR (Arabidopsis thaliana.org/, accessed on 15 September 2023) and Phytozome database (https://phytozome-next.jgi.doe.gov/, accessed on 15 September 2023). The multiple sequence alignment was performed using the ClustalW with the default settings. The phylogenetic tree based on neighbor-joining (NJ) method was constructed using MEGA-X software with bootstrap values from 1,000 replicates indicated at each node. The constructed phylogenetic tree was embellished using website ChiPlot (https://www.chiplot.online/, accessed on 20 September 2023).

Gene synteny analysis and MsSHMTs paralogs gene pair identification

One step MCScanX was employed to identify putative homologous chromosomal regions within and between M. sativa with the default parameters. Collinearity results within M. sativa genome were mapped using advanced circus software in TBtools. Multiple synteny plot was used to show the synteny relationship of the orthologous SHMT genes obtained from M. sativa, A. thaliana, and G. max [25].

Cis-elements in the upstream of MsSHMTs

The upstream 2000 bp sequences of the MsSHMTs coding region were retrieved from the M. sativa genomic database and submitted to PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html, accessed on 21 September 2023) to identify regulatory elements involved in hormone and stress responses. The result was visualized with ChiPlot (accessed on 21 September 2023).

Analysis of MsSHMTs expression pattern in different tissues

The RNA-seq data on different tissues of M. sativa were downloaded from the NCBI short read archive database as accession SRP055547 [26]. The genes corresponding to the XinJiangDaYe MsSHMTs were screened by online Blast (https://medicago.legumeinfo.org/, accessed on 2 October 2023). Expression data of six tissues including flowers, leaves, pre-elongating stems, elongating stems, roots, and nodules were normalized and visualized using the pheatmap R package. Genes with significantly higher expression levels in a specific tissue compared to others are considered tissue-specific genes (p < 0.05).

Plant materials, growth conditions, and treatments

Alfalfa (cultivar Gannong NO.3) was provided by the Gansu Agricultural University, Lanzhou, China. We selected full and consistent seeds, sterilized them with 6% NaClO for 5 min, evenly distributed the seeds onto vermiculite, and placed them in a photoperiodic growth chamber for cultivation. The temperature during cultivation should be maintained at 25/20℃ (day/night), with a 14/10 h (light/dark) cycle. 6 days after sowing, the M. sativa seedlings were transplanted into 1/2 Hoagland nutrient solution for hydroponics. When the seeds were 4 weeks of age, seedlings with uniform growth were treated with ABA (100 µM), SA (100 µM), MeJA (100 µM), drought (20% PEG6000), salt (200 mM NaCl), alkali (150 mM NaHCO3), and low (4 °C) and high (37 °C) temperatures. Samples were collected at 0 h (CK) and 3 h, 6 h, 12 h, 24 h, and 48 h after treatment. All sample collections were set up with three biological replicates. The samples were quickly frozen in liquid nitrogen after sampling, then transferred to -80 °C, and stored for subsequent analysis.

RNA extraction and real-time quantitative PCR

Total RNA was extracted from the samples using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The FastQuant First Strand cDNA Synthesis Kit (Tiangen, Beijing, China).

was used to synthesize cDNA. qRT PCR was carried out using the LightCycler 480 Real-Time PCR System (Roche Applied Science) and SYBR® Green Premix Pro Taq HS qPCR Kit. Three biological replicates with three technical replicates each were performed. The primers were designed by Primer 5.0 software and listed in Table 1. The reference gene is MsActin (MS.gene049321.t1). The ­2−∆∆CT calculation method was used to quantify the relative expression of the target gene.

Table 1 Primers used for qRT-PCR analysis
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Result

Identification and characterization of MsSHMTs in M. sativa

As shown in Table 2, a total of 15 MsSHMT genes were identified in the M. sativa genomes. Basic information analysis found that MsSHMTs are unevenly located on chromosomes. The protein length of the MsSHMTs ranges from 593 (MsSHMT5) to 99 (MsSHMT14) amino acids (aa), with corresponding molecular weights of 11.33 kDa and 65.98 kDa, respectively. Physicochemical property analysis showed that the isoelectric points of MsSHMTs are concentrated between 5.29 (MsSHMT15) and 9.35 (MsSHMT2), with most of them being neutral and basic proteins. The instability index analysis found that most of MsSHMT proteins are stable (Instability index < 40) except MsSHMT1, 5, and 11. In addition, 93% of MsSHMT proteins are hydrophilic (GRAVY < 0). Subcellular localization prediction revealed that MsSHMTs are distributed in the mitochondrial, Cytoplasmic, vacuole, and Chloroplast, where MsSHMT1/2/8/14/15 are cytoplasmic localization proteins, MsSHMT4/5/6/9/10 are chloroplast localization proteins, and MsSHMT11/12 are mitochondrial localization proteins.

Table 2 Basic information of 15 MsSHMTs identified in this study
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Phylogenetic analysis of SHMT gene family

To study the evolutionary history of the SHMT gene family in M. sativa, we constructed a phylogenetic tree from the alignment of 76 full-length SHMT protein sequences in model monocotyledon and dicotyledonous using the NJ method. The results revealed that SHMTs can be categorized into 4 distinct classes (Class I-IV), encompassing 21, 18, 15, and 22 proteins, respectively (Fig. 1). Notably, MsSHMT proteins are present in all classes. Class I consists of 5 TaSHMTs, 4 GmSHMTs, 3 PtrSHMTs, 2 MsSHMTs/AtSHMT/CsSHMTs/OsSHMTs, and 1 SlSHMT. Within Class II, MsSHMT10 is highly homologous to GmSHMT05c and GmSHMT08c. Class III contains 8 MsSHMHT members (MsSHMT2/3/4/6/7/8/14/15), which displayed the highest level of homology with GmSHMTs, AtSHMTs, and SlSHMTs, while OsSHMT proteins are absent. In Class IV, 4 members of the MsSHMT family, MsSHMT1/11/12/13, exhibit relatively close evolutionary relationships with GmSHMT02m, GmSHMT14m, GmSHMT08m, and GmSHMT18m.

Fig. 1
figure 1

Phylogenetic analysis of SHMT gene family. The amino acid sequences of SHMT proteins from O. sativa (5), T. aestivum (14), A. thaliana (7), G. max (13), C. sativus (6), S. lycopersicum (7), P. trichocarpa (9), and M. sativa (15) were used for analysis

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Synteny analysis of MsSHMT genes in M. sativa, Arabidpsis, and G. max

To explore the expansion patterns of M. sativa SHMT genes, we embarked on the relationship between M. sativa SHMT gene sequences. The analysis unveiled the occurrence of 3 fragment duplication event, MsSHMT3/6, MsSHMT3/7, and MsSHMT6/7. However, no occurrences of segmental duplication events were identified (Fig. 2A). The Ka/Ks values of these fragment duplication genes are lower than 1, suggesting that SHMT family genes evolved mainly under the influence of purifying selection (Supplementary Table S1). Through comparative analysis of the SHMT genes genetic relationship between M. sativa, A. thaliana, and G. max, we found 4 orthologous genomic fragments shared between M. sativa and A. thaliana, along with 11 orthologous fragments shared between M. sativa and G. max (Fig. 2B). Notably, chromosomes 2.1, 3.1, and 5.1 of M. sativa exhibit a higher frequency of homologous segments containing 3, 7, and 5 homologous genes, respectively.

Fig. 2
figure 2

Gene duplication and collinearity analysis of MsSHMTs. A: Schematic representation for the chromosomal distribution and interchromosomal relationships of MsSHMTs. Gray lines indicate all synteny blocks in the M. sativa genome, and the red line indicates segmental duplication MsSHMT gene pairs; B: collinear relationships of genes pairs from M. sativa, A. thaliana, and G. max. Gray lines in the background indicate the collinear blocks within M. sativa, A. thaliana, and G. max genomes and red lines indicate the collinear SHMT gene pairs

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Gene structure and conserved domain analysis of MsSHMT family genes

Based on the gene annotation files, we analyzed the exon-intron structural characteristics of MsSHMT genes. As illustrated in Fig. 3A, the number of exons vary in the range of 3 to 15. Among them, MsSHMT11/12 manifest 15 exons, MsSHMT2/3/6/13 manifest 10 exons, MsSHMT5/8/9/10 exhibit 4 exons. MsSHMT genes demonstrating highly similar exon-intron structures were clustered within the same clade, for instance, MsSHMT5/9/10 and MsSHMT3/6/7. The investigation of conserved motifs revealed the presence of 10 conserved motifs in the MsSHMTs, with lengths spanning from 11 to 50 amino acids (Supplementary Figure S1). Conserved motifs among the MsSHMTs within a particular clade present a marked resemblance. Nevertheless, certain genes exhibit significant variations in motif composition within the same clade, such as MsSHMT8 and MsSHMT14 (Fig. 3B).

Fig. 3
figure 3

Gene structure and conserved domain analysis of MsSHMTs in M. sativa. A: Exon-intron structure of MsSHMTs. B: Motif distribution of MsSHMT proteins. Different motifs (1–10) are indicated by different colors. The sequence logos and information for each motif are provided in Additional files Figure S1

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Cis‑acting elements in the MsSHMTs promoters

To explore the potential functions of MsSHMTs, we analyzed the gene’s promoter region sequence. The results revealed a significant presence of cis-acting elements in MsSHMTs, which can be categorized into two groups: hormone-related and stress-related elements (Fig. 4A). Hormone-associated elements include the ABA-responsive element (ABRE), MeJA-responsive element (CGTCA/TGACG-motif), SA-responsive element (TCA-element), IAA-responsive element (TGA), and GA-responsive element (P-box). Stress-associated elements include TC-rich repeat sequences, a drought response element (MBS), a low-temperature response element (LTR), an anaerobic response element (ARE), and a wound-responsive element (WUN-motif). Certain elements are shared among multiple MsSHMTs (Fig. 4B). For instance, all MsSHMTs except MsSHMT2/6/9/13 contain ABRE elements. CGTCA-motif and TGACG-motif are present in all MsSHMTs except MsSHMT6. ARE motifs are found in all MsSHMTs except MsSHMT6.

Fig. 4
figure 4

Identification of the cis-acting element in the 2-kb promoter region of MsSHMT genes. (A) Statistics on the number of cis-acting elements of MsSHMTs. (B) Analysis of shared cis-acting elements of MsSHMTs. The numbers on the box represent the count of cis-elements in the promoter

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Tissue expression profile analysis of MsSHMT genes

To research the role of MsSHMTs in the growth and development of M. sativa, we analyzed the expression patterns of the MsSHMT genes in various tissues using published transcriptome data. The results indicated that the MsSHMTs are expresses in all tissues of M. sativa (Fig. 5). However, the expression patterns vary among MsSHMTs. In nodules, the expression levels of MsSHMT4/6/7/14 were the highest. MsSHMT1/3/9/11 showed high expression in leaves, followed by stems. The expression level of MsSHMT2 in roots was significantly higher compared to other tissues. MsSHMT5 exhibited high expression in both roots and flowers. MsSHMT8/10/12/13/15 displayed high expression levels in both roots and stems. These findings suggest that MsSHMTs may play a significant role in the developmental processes of M. sativa leaves, roots, and nodules.

Fig. 5
figure 5

Expression patterns of MsSHMTs in different tissues of M. sativa. The data of transcriptional level comes from the NCBI (accession number: SRP055547). Red and blue represent high and low expression, respectively

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Expression profile analysis of MsSHMT genes under hormones

To explore the response patterns of MsSHMT members to phytohormones, qRT-PCR was employed to detect the expression profiles of 15 MsSHMTs under ABA, SA, and MeJA treatments. The results indicate that transcript levels of all MsSHMTs exhibited an initial increase followed by a decrease under ABA treatment (Fig. 6A). Among them, MsSHMT1/2/10/11/12/13 reached their highest expression level at 3 h, while MsSHMT3/6/7/8/9/15 peaked at 6 h. Additionally, MsSHMT5/4/14 demonstrated higher expression levels at 6–12 h. Under SA treatment, the expression levels of MsSHMT1/2/6/9/11/12/14 were repressed (Fig. 6B). However, other MsSHMT members including MsSHMT4/8/13/10 were up-regulated, reaching their peak expression level at 24 h, and MsSHMT3/5/7/15 reached their highest expression level at 48 h. Under MeJA treatment, the expression levels of most MsSHMT members were inhibited, such as MsSHMT1/2/3/6/10/14. However, other members such as MsSHMT5/15 were up-regulated at 3 h, and MsSHMT4/7/13 were up-regulated at 24 and 48 h (Fig. 6C).

Fig. 6
figure 6

Expression levels of MsSHMT genes under ABA (A), SA (B) and MeJA (C) treatment. The color scale represents the folding changes normalized by log2 transformed data. Blue represents down-regulated genes and red represents up-regulated genes

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Fig. 7
figure 7

qRT-PCR analysis of MsSHMT genes in response to salinity, alkali, drought, cold, and heat treatments. All data were expressed as the mean ± standard error (SE) of three independent replicates. Duncan’s analytical test (P < 0.05) was used to determine the significance of the differences between 0 h

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Expression profile analysis of MsSHMT genes under abiotic stresses

Upon further analysis, it was found that the expression levels of MsSHMT genes vary under various abiotic treatments. Regarding MsSHMT1, its expression level is down-regulated under alkali and cold treatment, while it is significantly up-regulated under salt, heat, and drought treatments (Fig. 7). In the case of MsSHMT2, there is almost no change in expression level under drought and cold treatment, but it is significantly up-regulated under salt, alkali, and heat treatments. MsSHMT3/5/13 are up-regulated under salt and drought treatments, but remain almost unchanged under cold treatment. Meanwhile, MsSHMT8 is up-regulated by drought treatment, while its expression is down-regulated under other treatments. MsSHMT4 is up-regulated under cold and heat treatments, reaching its highest expression level at 12 h. MsSHMT6/7/9 are up-regulated under drought treatment, reaching their highest expression levels at 24 h, 12 h, and 3 h, respectively. MsSHMT10 is up-regulated under drought treatment, reaching a higher expression level at 3 h, but remains almost unchanged under salt and alkali treatments. MsSHMT11 is significantly induced by cold stress. MsSHMT14 is up-regulated under salt and drought treatments, but remains almost unchanged under heat treatment. There is down-regulation of MsSHMT15 expression level under salt treatment, while it is significantly up-regulated under alkali and drought treatments.

Discussion

With the release of the M. sativa (Zhongmu No. 1 and Xinjiang Daye) genomes, a large number of genes related to stress response and growth development have been explored [21, 27]. Serine hydroxymethyltransferase (SHMT), as an important enzyme shared by both animals and plants, has garnered significant attention from researchers in the field of plant biology. Studies have reported that G. max contains 18 members of the SHMT gene family, T. aestivum has 14, P. trichocarpa has 9, A. thaliana has 7, S. lycopersicum has 7, and O. sativa has 5 [7, 10, 13, 20, 28, 29]. In this study, we identified a total of 15 members of the SHMTs family in the genome of M. sativa (Table 1). This count is close than that of G. max and T. aestivum but greater than that of A. thaliana, S. lycopersicum, and O. sativa. It appears to be correlated with genome size and the number of whole-genome duplications. Further analysis of the replication event of MsSHMTs genes revealed the presence of 3 pair fragment duplication genes (MsSHMT3/6, MsSHMT3/7, and MsSHMT6/7) with a Ka/Ks value of less than 1 (Table S1). This indicates that MsSHMTs have undergone purifying selection. Comparison of collinearity relationships between M. sativa, G. max, and A. thaliana revealed that one MsSHMT gene corresponds to multiple GmSHMT genes, suggesting an independent replication event in G. max (Fig. 2) [30]. These results suggest that the expansion of MsSHMTs is primarily driven by whole-genome duplication and segmental duplication, consistent with previous research in G. max, T. aestivum, and S. lycopersicum [29].

Evolutionary analysis plays a crucial role in revealing the evolutionary history of gene families, determining orthologous genes, and inferring gene functions, among other aspects [31]. Previous studies have shown that SHMTs can be classified into 4 subclasses based on their location within different organelles: mitochondria, chloroplasts, nucleus, and cytoplasm [32]. This classification is consistent with the findings of this study, where 76 SHMT proteins were classed into 4 subclasses (Fig. 1). However, whether these subclasses are associated with distinct subcellular localization of the proteins still requires extensive experimental validation. Furthermore, SHMT activity has been detected in other organelles such as the endoplasmic reticulum [20]. Surprisingly, it has been revealed that a specific SHMT gene loss event occurred in some monocotyledonous [29]. In this study, the absence of Class III SHMT in monocotyledonous O. sativa, and whole T. aestivum did not exhibit this loss in the SHMT family. This discrepancy may be attributed to the reduced necessity of Class III SHMT genes in O. sativa due to long-term genetic evolution. Alternatively, it could be a result of the natural degradation after an accidental gene duplication event, where redundant copies are no longer retained through natural selection [33]. Furthermore, the genes belonging to the same clade exhibit a high similarity in both gene structure and motif composition, as exemplified by MsSHMT5/9. However, a subset of genes, such as MsSHMT8 and MsSHMT14 displayed significant differences in gene structure. This finding suggests that SHMTs are evolutionarily conserved but undergo adaptive structural changes [34]. The number of exons among members of the M. sativa SHMT family varies from 3 to 15, indicating MsSHMT family maybe experience exon retention or alternative splicing events during evolution [35].

In plants, SHMT genes have been identified in numerous tissues [28]. In A. thaliana, AtSHMT1 is predominantly found in leaves, stems, and flowers, while AtSHMT3 is primarily expressed in germinating seeds, and the transcript accumulation of AtSHMT4 is restricted to the roots of seedlings [36]. BvSHMTa is expressed in both leaves and roots of sugar beets [37]. In G. maxs, most GmSHMT genes exhibit ubiquitous expression across all tissues [29]. This is consistent with results in M. sativa suggesting that the diversification of spatiotemporal expression patterns may represent a common evolutionary feature of the SHMT gene family, enabling SHMTs to function across various tissues at different growth and developmental stages [38]. Among all the analyzed tissues, the expression of MsSHMT1/3/9/11 was most highly expressed in leaves, while transcripts of MsSHMT4/6/7/14 were most expressed in root nodules, and the transcription levels of MsSHMT2/5/8/13/15 were highly expressed in roots (Fig. 5). These findings indicate that MsSHMT genes may play pivotal roles in processes such as leaf photosynthesis, root system stability, and nitrogen fixation in root nodules [39].

The response of SHMTs to hormones has been reported in numerous species [12]. In our study, the MsSHMT gene exhibited responses to ABA, SA, and MeJA treatments, suggesting its involvement in hormone regulation and consequently, in controlling the growth and development of M. sativa [40]. Additionally, this transcriptional activation may be associated with multiple hormone-responsive elements in the promoter region of the MsSHMT genes [41]. However, our further investigations have yielded some conflicting results. For instance, while MeJA response elements were found in all MsSHMT genes, most genes did not show significant induction under MeJA treatment (Fig. 6). This may be attributed to the fact that the regulation of gene expression is not solely reliant on a single factor but rather involves the collaborative action of multiple transcription factors. Moreover, the limited availability of tissue, growth stages, and treatments may influence the interpretation of their relationships. Therefore, further experiments are necessary to elucidate the relationship between hormone responses and cis-acting elements.

As vital regulators of plant growth and development, SHMT gene family have been reported to respond to adverse environment in numerous species [7, 10, 29]. In A. thaliana, AtSHMT1 collaborates with UBP16 (ubiquitin-specific protease) to control plasma membrane Na+/H+ antiporter activity under salt stress, thereby reducing Na+ accumulation and enhancing plant tolerance to salt stress [42]. Overexpression of OsSHMT promotes scavenging of excessive harmful H2O2 by interacting with APX, Hsp70, ATP-synα, ATP-synβ, and MSCP, thus enhancing cold tolerance in O. sativa [43]. Additionally, studies have found that plant SHMT mitigates damage caused by low temperatures and drought by maintaining redox homeostasis and regulating stomatal closure [6]. In our study, MsSHMT genes responded to various stress stimuli consisting with the findings in S. lycopersicum SHMT research [7]. Specifically, MsSHMT1/2/3/5/11/12 were significantly induced by salt stress; MsSHMT2/4/13/15 were markedly induced by alkaline stress; while the expression levels of MsSHMT1/3/6/7/8/15 were significantly upregulated under drought stress. Moreover, MsSHMT4 exhibited sensitivity to high and low temperatures (Fig. 7). This indicates functional redundancy of MsSHMTs in mediating M. sativa responses to abiotic stresses. Considering these diverse stress response patterns, we hypothesize that the functional differences among these genes may be associated with cis-acting elements in their promoter regions, such as TC-rich repeat sequences, drought response elements (MBS), and low-temperature response elements (LTR). The presence of these cis-acting elements is likely responsible for the differential expression patterns of various MsSHMTs under specific stress conditions, implying their importance in regulating MsSHMT gene responses to environmental stresses. The differences in functionality among gene family members may be attributed to adaptive evolution. The functions of MsSHMT genes need to be further investigated in depth, and Fig. 8 shows a potential regulatory model of MsSHMTs.

Fig. 8
figure 8

A working model of MsSHMTs response to hormones and abiotic stresses. Under abiotic stress, salt stress leads to a sudden increase in intracellular Na+ content, and intracellular calcium signaling as well as SOS signaling are activated to increase the expression of genes such as MsSHMT2/6/13 by binding to their promoters to mediate M. sativa response to abiotic stress. And hormone-responsive genes such as MsSHMT1/3/15 may act in the regulation of M. sativa growth and development through transcription factors that bind to related cis-acting elements in the promoter regions of the genes

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Conclusion

In general, 15 SHMT genes were identified in the M. sativa genome in this study. Physicochemical properties, gene and protein structures, and evolutionary relationships revealed structural and functional similarities and differences in M. sativa SHMTs. Tissue-specific analyses indicated that MsSHMT members play key housekeeping functions by regulating cellular metabolism during plant growth and development. In addition, M. sativa SHMTs are involved in hormonal responses and alleviating various abiotic stresses, especially salt stress and drought stress. In summary, this study aims to provide support for further studies on the involvement of the MsSHMT gene family in M. sativa growth regulation and stress response, and to provide a theoretical basis for further exploration of the functions of plant SHMT members.

Data availability

All data generated or analyzed in this study are included in this published article and its supplementary material. The draft genome data of autotetraploid cultivated (‘Xinjiang Daye’) alfalfa was obtained from the figshare projects (https://figshare.com/projects/whole_genome_sequencing_and_assembly_of_Medicago_sativa/66380). Genome-wide transcriptome data of different alfalfa tissues were acquired from the NCBI short read archive database as accession SRP055547.

Abbreviations

SHMT:

Serine hydroxymethyltransferase

GDC:

Glycine Decarboxylase Complex

ROS:

Reactive oxygen species

SCN:

Soybean cyst nematode

ER:

Endoplasmic reticulum

Hsp70:

Heat shock protein 70

HMM:

Hidden Markov Model

qRT-PCR:

Quantitative reverse transcription polymerase chain reaction

Ka/Ks:

Nonsynonymous substitution rate/synonymous substitution rate

FPKM:

Fragments Per Kilobase of transcript per Million mapped reads

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Acknowledgements

We are grateful to members of our laboratory for helpful criticism and advice.

Funding

This research was supported by the alfalfa breeding project (2022ZD0401102).

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  1. College of Pratacultural Science, Key Laboratory of Grassland Ecosystem, Ministry of Education, Pratacultural Engineering Laboratory of Gansu Province, Sino-U.S. Center for Grazingland Ecosystem Sustainability, Gansu Agricultural University (36.0° N, 103 8° E), Yingmencun, Anning District, Gansu province, Lanzhou, Gansu, 730070, China

    Rong Gao, Lijuan Chen, Fenqi Chen & Huiling Ma

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HLM and RG conceived and designed the experiment. RG and FQC performed the experiments. RG and LJC analyzed all the data. RG wrote he manuscript. HLM revised the manuscript. All of the authors read and approved the final manuscript.

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Gao, R., Chen, L., Chen, F. et al. Genome-wide identification of SHMT family genes in alfalfa (Medicago sativa) and its functional analyses under various abiotic stresses. BMC Genomics 25, 781 (2024). https://doi.org/10.1186/s12864-024-10637-z

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BMC Genomics

ISSN: 1471-2164

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