Genome-Wide Identification and Expression Profiling Analysis of the Trihelix Gene Family Under Abiotic Stresses in Medicago truncatula.

The trihelix transcription factor (GT) family is widely involved in regulating plant growth and development, and most importantly, responding to various abiotic stresses. Our study first reported the genome-wide identification and analysis of GT family genes in Medicago truncatula. Overall, 38 trihelix genes were identified in the M. truncatula genome and were classified into five subfamilies (GT-1, GT-2, SH4, GTγ and SIP1). We systematically analyzed the phylogenetic relationship, chromosomal distribution, tandem and segmental duplication events, gene structures and conserved motifs of MtGTs. Syntenic analysis revealed that trihelix family genes in M. truncatula had the most collinearity relationship with those in soybean followed by alfalfa, but very little collinearity with those in the maize and rice. Additionally, tissue-specific expression analysis of trihelix family genes suggested that they played various roles in the growth and development of specific tissues in M. truncatula. Moreover, the expression of some MtGT genes, such as MtGT19, MtGT20, MtGT22, and MtGT33, was dramatically induced by drought, salt, and ABA treatments, illustrating their vital roles in response to abiotic stresses. These findings are helpful for improving the comprehensive understanding of trihelix family; additionally, the study provides candidate genes for achieving the genetic improvement of stress resistance in legumes.

1. Introduction

Transcription factors (TFs) are a kind of DNA binding protein, that play pivotal roles in plant growth and development, as well as in response to environmental stresses [1,2]. TFs regulate the expression of target genes by binding to specific cis-elements of the gene promoter region or binding with other TFs [3]. Currently, trihelix TF family members have attracted more attention; they feature a typical helix–loop–helix–loop–helix structure with a core sequence of 5′-G-Pu-(T/A)-A-A-(T/A)-3′ in their DNA-binding domain [4,5]. Because this domain can specifically bind to GT elements required for light response in a DNA sequence, it is also called the GT family [6]. The conserved domain of trihelix is similar to the individual repeats of the MYB family in sequence; therefore, it is generally thought to be derived from MYB-like genes [5].

The first discovered trihelix gene was the GT-1 transcription factor in pea (Pisum sativum) [7]. Subsequently, orthologous genes were identified in tobacco and Arabidopsis thaliana [8,9]. Early researches on GT genes had been focused on the light response regulation [10,11,12]. In recent years, the biological functions of several trihelix family genes have been discovered, indicating that they are widely involved in fine-tuning a range of specialized developmental processes including flowers, trichomes, stomata, embryos and seeds, and most importantly, responding to various abiotic stresses in plants [12,13,14,15,16,17,18]. Based on the phylogenetic relationship and characteristics of the conserved domain, the trihelix genes family is divided into five subfamilies: GT-1, GT-2, GTγ, SH4, and SIP1. Park et al. found that AtGT-3b was rapidly induced by salt stress and could interact with the GT-1 cis-element of the SCaM-4 promoter in Glycine max, thereby responding to various environmental stresses [18]. The GT-2 LIKE 1 (AtGTL1) can improve water use efficiency and drought tolerance by trans-repressing the expression of the STOMATAL DENSITY AND DISTRIBUTION1 (SDD1) gene, encoding a subtilisin-like serine protease that negatively regulates stomatal generation in Arabidopsis [15]. Also in wheat, TaGT2L1D, the homologous gene of AtGTL1, plays a similar role and affects floral organ development [19]. The expression levels of three GTγ clade genes in rice (OsGTγ-1, OsGTγ-2, and OsGTγ-3) increased significantly under salt stress; moreover, the OsGTγ-1 gene could respond to drought and cold stress [20]. Lin et al. demonstrated that Shattering1 (SHA1), encoding a member of the trihelix SH4 subfamily, plays an important role in the activation of cell separation in the shattering process of rice seeds [17].

M. truncatula is a diploid plant with a relatively small genome, adopted as a model species for legumes genetics and functional genomics research [21,22]. Particularly, its genome has a high similarity to that of alfalfa (Medicago sativa), the most widely cultivated economic forage plant in the world; hence, studies on M. truncatula can provide important genetic reference information for alfalfa. At present, trihelix genes have been systematically identified and analyzed in many plants, including Arabidopsis [12], Oryza sativa [23], Solanum lycopersicum [24], Brassica rapa [25], Gossypium arboreum [26], G. max [27], Chrysanthemum morifolium [28], Triticum aestivum [29] and Populus trichocarpa [30]. However, information and functional analysis of the trihelix family in M. truncatula has not yet been performed. Because trihelix family genes are widely involved in plant growth and development and respond to various abiotic stresses, identifying and analyzing the expression characteristics of trihelix family members in M. truncatula is of great significance.

In this study, we performed a systematic genome-wide investigation of the trihelix gene family in M. truncatula. Overall, 38 MtGT genes were identified, and their phylogenetic relationship, chromosomal localization, gene structures, and motif compositions were analyzed in detail. We demonstrated the collinearity of MtGT genes with A. thaliana, G. max, Zea mays, O. sativa and M. sativa genomes. Additionally, we performed tissue-specific expression analysis of MtGT genes in six tissues (blade, bud, nodule, flower, root, and seedpod) and verified by real-time quantitative PCR (qRT-PCR). Moreover, we explored and validated the expression profiling of MtGT genes in response to drought and salt stresses. Therefore, this study is helpful to understand the trihelix family more comprehensively and provides a reference functional gene resource, particularly for alfalfa, for the genetic improvement of stress resistance in legumes.

2. Materials and Methods

2.1. Plant Materials and Treatments

M. truncatula (cv. Jemalong A17) seeds were sterilized in 75% ethanol for 5 min, rinsed with sterile water five times, and then placed on the moistened filter paper in Petri dishes. They were subsequently cultured in a growth cabinet at 25 °C. For the tissue-specific expression analysis of MtGT genes, 7-day-old seedlings were transferred into the mixture of peat soil and vermiculite (1:1, v/v) for individual pot cultivation (18 cm inner diameter, 20 cm height). Potted seedlings grew in a greenhouse (the temperature is 25 °C with a 16/8 h light/dark photoperiod, relative humidity of 35–40%, and photon flux density of 450 μmol m−2·s−1). Roots, stems, blades, buds, flowers and seedpods were collected at pod stage from three individual plants. For the expression analysis of MtGT genes response to different treatments, the 7-day-old seedlings were transferred into the flasks with 1/2 MS liquid medium and grew in a controlled growth chamber under 16/8 h light/dark regime at 25 °C. Ten days later, the plants with the fourth blade expanded were watered with 15% PEG6000, 200 mmol·L−1 NaCl, and 1 mmol·L−1 ABA, respectively. The blades were collected at 0 h, 2 h, 24 h and 72 h with a triplicate. All samples were immediately frozen in liquid nitrogen and stored at −80 °C for RNA extraction.

2.2. Identification of the Trihelix Genes in M. truncatula

The M. truncatula genome information was downloaded from M. truncatula Genome Database (MTGD, http://www.medicagogenome.org/downloads). Based on two BLASTp methods, the 35 trihelix protein sequences of Arabidopsis retrieved from Plant Transcription Factor Database (PlantTFDB, http://planttfdb.cbi.pku.edu.cn/) [31] were used as query to obtain the possible trihelix proteins in M. truncatula genome by BLASTp search with a cutoff E-value of 1.0 × 10−10. Furthermore, the Hidden Markov Model (HMM) profile (PF13837) was used to identify the putative trihelix domain in Pfam database (http://pfam.xfam.org/) [32]. The Conserved Domain Database (CDD) of NCBI (https://www.ncbi.nlm.nih.gov/cdd/) [33] and SMART (http://smart.embl-heidelberg.de) [34] were used to confirm the trihelix proteins in all the candidate proteins. The basic physical and chemical characteristics of 38 trihelix proteins, such as the molecular weight (MW), isoelectric point (pI) and grand average of hydropathicity (GRAVY), were determined on ExPASy (http://web.expasy.org/protparam/) [35]. The subcellular localization of MtGTs were predicted on the Plant-mPLoc website (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/) [36].

2.3. Chromosomal Distribution and Gene Duplication Events Analysis

The chromosomal location information of 38 trihelix genes were obtained from the M. truncatula genomic annotation file GFF3 (general feature format) that was downloaded in MTGD. The TBtools software [37] was used to draw the chromosomal distribution image of MtGT genes. The detection and identification of the gene duplication events in MtGT genes were performed using multiple collinear scanning toolkits (MCScanX) [38] with E-value set to 10−5.

2.4. Gene Structure and Conserved Motifs Analysis

Gene structure was constructed to visualize the exon-intron of MtGT genes based on the CDS and the corresponding full-length sequence by using TBtools software [37]. The MEME tool (http://meme-suite.org/tools/meme) [39] was used to analyze the conserved motifs of MtGT proteins and the relative parameters were set to the motif width as 15–50 amino acid (aa) and the number of motifs as 10.

2.5. Phylogenetic and Collinearity Analysis of MtGT genes

The full-length amino acid sequences of the M. truncatula and Arabidopsis trihelix family proteins were aligned using MUSCLE, and were visualized by Jalview 2 [40]. The MEGA X [41] was used to construct the unrooted phylogenetic tree by Neighbor-Joining (NJ) method with a bootstrap value of 1000 replicates. The phylogenetic tree was illustrated using online tool EvolView (http://www.evolgenius.info/evolview/) [42]. To analyze syntenic relationships of the trihelix family genes among the M. truncatula and Arabidopsis, G. max, Z. mays, O. sativa and M. sativa genomes, MCScanX was used with default settings [38] and was visualized by TBtools software [37].

2.6. Tissue-Specific Expression and Abiotic Stress Expression Analysis

The relative FPKM value (fragments per kilobase of transcript per million fragments mapped) of 36 MtGT genes in six tissues (blade, bud, nodule, flower, root, seedpod) of RNA-seq data were retrieved from MTGD (http://www.medicagogenome.org/) and the heat map of hierarchical clustering was visualized using TBtools software [37]. 33 MtGT genes expression microarray data of 28-day-old seedlings under drought (40–45% soil water content) and salt (200 mM NaCl) treatments were obtained from the M. truncatula Gene Expression Atlas (MtGEA) (https://mtgea.noble.org/v3/) Web Server [43], and visualized for heat map using TBtools software [37].

2.7. Expression Analysis of the MtGT Genes by Real-Time qPCR

Total RNAs were extracted with the Eastep® Super Total RNA Extraction Kit (Promega, Beijing, China) following the manufacturer’s instructions and then EasyScript® All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (TransGen, Beijing, China) was used to synthesize the cDNA. Quantitative real-time PCR (qRT-PCR) was conducted according to the instructions of 2×RealStar Green Fast Mixture with ROX II (GenStar, Beijing, China) on an ABI QuantStudioTM 7 Flex RT-PCR system (Applied Biosystems, Foster City, CA, USA). The gene-specific primer sequences for qRT-PCR determination are provided in Table S9. MtActin was used as the internal control. Three technical repetitions for each sample, and the relative expression data were calculated according to the 2−ΔΔCT method [44].

3. Results

3.1. Identification of MtGT Genes in M. truncatula

In this study, 38 non-redundant trihelix genes were identified in the M. truncatula genome through two BLAST methods based on the known trihelix protein sequences of Arabidopsis, and both Pfam and CDD databases confirmed the presence of trihelix domain. Subsequently, 38 MtGT genes were named MtGT1 to MtGT38 according to their order on the chromosomes (Table 1). The predicted physical and chemical properties of MtGT proteins, including protein length, molecular weight (MW), isoelectric point (pI), and grand average of hydropathicity (GRAVY) are shown in Table 1. The length of MtGT proteins varied from 189 (MtGT-2) to 1223 (MtGT-15) amino acids (aa), with a MW range of 21.31–140.86 kDa. The average length of these proteins was 428 aa, and their average MW was 48.69 kDa. The pI ranged from 4.48 (MtGT-17) to 9.73 (MtGT-24). The GRAVY ranged from −0.400 (MtGT-19) to −1.395 (MtGT-17), and the average GRAVY was 0.953. Subcellular localization prediction revealed that most MtGT proteins are located in the nucleus, which is consistent with the role of TFs. Interestingly, the remaining four proteins (MtGT-11, MtGT-12, MtGT-13, and MtGT-20) are located in the chloroplast, which may be related to photosynthesis. The nucleic acid sequences of MtGT genes and encoded amino acid sequences are provided in Table S1.

Table 1

The related information of trihelix genes in M. truncatula.

Name	Gene ID	Length (aa)	Signature Domain	MW (KDa)	Subcellular Localization	pI	GRAVY
MtGT-1	Medtr1g009220.1	319	19–104	37,270.73	Nucleus	5.36	−1.039
MtGT-2	Medtr1g037650.1	189	35–122	21,313.82	Nucleus	9.14	−0.917
MtGT-3	Medtr1g060990.1	511	129–217	58,779.54	Nucleus	6.57	−1.173
MtGT-4	Medtr1g061640.1	496	121–247	56,941.93	Nucleus	6.46	−1.242
MtGT-5	Medtr1g081180.1	557	101–181	64,373.39	Nucleus	5.71	−1.104
MtGT-6	Medtr1g492790.1	501	112–200	57,848.24	Nucleus	5.99	−0.921
MtGT-7	Medtr1g094045.1	381	53–183	42,978.72	Extracell/Nucleus	9.09	−1.072
MtGT-8	Medtr1g098870.1	565	53–140	64,378.61	Nucleus	5.91	−1.088
MtGT-9	Medtr1g098900.1	637	55–142	70,838.09	Nucleus	5.77	−0.900
MtGT-10	Medtr1g098920.1	450	123–250	51,386.17	Nucleus	6.16	−1.049
MtGT-11	Medtr1g112650.1	316	40–131	35,249.58	Chloroplast/Nucleus	4.97	−0.653
MtGT-12	Medtr2g016140.1	332	20–114	37,437.82	Chloroplast	5.37	−0.703
MtGT-13	Medtr2g016140.2	317	20–114	35,816.88	Chloroplast/Nucleus	5.29	−0.805
MtGT-14	Medtr2g026725.1	379	260–348	42,411.51	Nucleus	7.74	−0.924
MtGT-15	Medtr2g092960.1	1223	43–136	140,855.87	Nucleus	8.51	−0.845
MtGT-16	Medtr2g098080.1	445	112–238	50,604.51	Nucleus	6.21	−0.991
MtGT-17	Medtr2g102227.1	496	272–360	57,303.69	Nucleus	4.48	−1.395
MtGT-18	Medtr3g014280.1	325	35–122	37,084.54	Nucleus	9.03	−1.057
MtGT-19	Medtr3g089020.1	867	773–857	96,355.08	Chloroplast	7.97	−0.400
MtGT-20	Medtr3g085960.1	390	74–163	44,613.12	Chloroplast/Nucleus/Peroxisome	5.90	−0.834
MtGT-21	Medtr4g015680.1	279	40–125	33,950.09	Nucleus	6.55	−1.283
MtGT-22	Medtr4g079960.1	313	28–123	35,566.23	Nucleus	9.53	−0.778
MtGT-23	Medtr4g079960.2	304	28–123	34,456.87	Nucleus	9.60	−0.818
MtGT-24	Medtr4g079960.3	315	28–123	35,784.64	Nucleus	9.73	−0.774
MtGT-25	Medtr4g117990.1	340	41–135	37,889.76	Nucleus	9.07	−0.915
MtGT-26	Medtr5g017500.1	229	20–115	26,637.29	Nucleus	9.22	−0.834
MtGT-27	Medtr5g026540.1	302	43–128	35,678.99	Nucleus	8.34	−1.208
MtGT-28	Medtr6g035315.1	526	117–243	59,762.71	Nucleus	6.05	−0.988
MtGT-29	Medtr6g035370.1	646	53–140	72,115.32	Nucleus	5.74	−1.018
MtGT-30	Medtr6g486270.1	370	127–218	42,750.57	Nucleus	4.59	−1.147
MtGT-31	Medtr7g020870.1	341	38–130	37,377.48	Nucleus	5.32	−0.740
MtGT-32	Medtr7g020870.2	245	38–131	26,785.93	Nucleus	5.77	−0.636
MtGT-33	Medtr7g068770.1	293	51–136	35,338.67	Nucleus	6.62	−1.346
MtGT-34	Medtr7g081190.1	388	43–140	43,393.75	Nucleus	9.64	−0.969
MtGT-35	Medtr7g103390.1	455	101–227	52,293.03	Nucleus	6.02	−1.204
MtGT-36	Medtr7g114860.1	483	379–476	55,781.34	Nucleus	6.34	−0.832
MtGT-37	Medtr8g022290.1	371	65–161	40,319.8	Nucleus	9.53	−0.803
MtGT-38	Medtr8g100130.1	361	45–139	40,525.42	Nucleus	5.36	−0.803

Length: Protein length (aa); MW: Protein molecular weight (kDa); pI: isoelectric point; GRAVY: Grand average of hydropathicity.

3.2. Phylogenetic Analysis and Classification of MtGT Genes

To investigate the molecular evolution and phylogenetic relationship of the trihelix family in M. truncatula, multiple sequences alignment of 35 A. thaliana trihelix proteins [12,31] and 38 MtGT proteins was performed (Figure S1) and the unrooted phylogenetic tree was constructed (Figure 1). A total of 73 trihelix proteins were classified into five clades (GT-1, GT-2, SH4, GTγ and SIP1), consistent with the previous studies on Arabidopsis [14] and other species, such as soybean [24], rice [17], tomato [21], and wheat [26]. Among these, SIP1 with 14 MtGT family members was the largest cluster, whereas GT-1 and GTγ were the smallest subfamily with 5 MtGT family members each. GT-2 and SH4 clades contained eight and six MtGT members, respectively. These results were similar to the genes distribution of different subgroups in Arabidopsis and rice [12,23].

Figure 1

Phylogenetic tree of trihelix proteins in M. truncatula and A. thaliana. The branches with different colors represent different subfamilies. The trihelix proteins of M. truncatula and A. thaliana are marked as red triangles and blue circles, respectively.

3.3. Chromosomal Distribution and Gene Duplication Events of MtGT Family

As shown in the chromosome map (Figure 2), 38 MtGT genes were located unevenly on eight chromosomes. Chromosome 1 contained most genes (11) of trihelix family, whereas chromosomes 5 and 8 had the least number of genes (2). To clarify the molecular evolution of the MtGT family, gene duplication events including tandem and segmental duplication were analyzed. In this study, we identified three groups of MtGT genes with tandem duplication events (MtGT-12, MtGT-13; MtGT-22, MtGT-23, MtGT-24; and MtGT-31, MtGT-32), and segmental duplication existed in two pairs of genes (MtGT-4, MtGT-35 and MtGT-5, MtGT-36) (Figure 2). Tandem and segmental duplication events facilitated divergence of genes with novel functions, which play a key role in promoting the evolution and expansion of gene families to help plants in adapting to various environmental conditions [25,45].

Figure 2

Chromosomal distribution and duplication events of MtGT genes. The chromosome number is labelled on the left of each chromosome. Red boxes indicate tandem duplication, and red lines indicate segmental duplication.

3.4. Gene Structural Characteristics and Conserved Motifs Compositions of MtGT Genes

The features of the MtGT genes structure are shown in Figure 3b, including the distribution of exons and introns and the types of protein domains. The most M. truncatula trihelix genes grouped in the same subfamily shared similar exon/intron organizations and domains. All MtGT genes contained the GT domain or MYB DNA-binding domain, in which members belonging to the SIP1 or GTγ clade contained the MYB DNA-binding domain, and most MtGT genes in GT-1, GT-2, and SH4 clades possessed the GT domain. Further, the majority of MtGT family members had very few introns (0–2) and 14 of them had no intron. The GTγ subfamily members (MtGT4, MtGT10, MtGT16, and MtGT35) had no intron except in MtGT28, which coincided with the findings of previous studies of trihelix families in B. rapa [25], wheat [29] and Fagopyrum tataricum [46]. In the remaining genes, MtGT19 had most introns (16), MtGT-15, which was the longest trihelix gene, had 11 introns, and MtGT20 contained four introns (Figure 3b). To further analyze the diversity of MtGT proteins, the MEME search tool was used to identify 10 conserved motifs (motif 1–motif 10) shown in Figure 3c, and the detailed sequence of each motif is provided in Table S2. Motif 1 was presented in all MtGT proteins. Motif 5 existed almost in all GT-1, GT-2, and SH4 clades. Among them, MtGT-5, MtGT-6, MtGT-8, MtGT-9, MtGT-29, MtGT-31, MtGT-32, and MtGT-36 had two motif 5, of which most belonged to clade GT-2. In addition, GTγ clade genes featured motif 9 at their N-terminal, and all SIP1 subfamily genes contained motif 2 at their N-terminal and featured motif 6 at C-terminal.

Figure 3

Gene structures and conserved motifs of the MtGT genes. (a) The phylogenetic tree was constructed based on the amino acid sequences of M. truncatula trihelix proteins. (b) Gene structures of M. truncatula trihelix genes. Green boxes indicate untranslated 5′- and 3′-regions, yellow boxes indicate exons, and gray lines indicate introns. The trihelix domains are labelled with dark blue boxes (GT1) and pink boxes (MYB DNA-binding). (c) The motif compositions of M. truncatula trihelix proteins. The motif 1-motif 10 are displayed in different colored boxes. The protein length can be estimated using the scale at the bottom.

3.5. Evolutionary and Collinearity Analysis within MtGT Genes and Several Species

To further understand the collinearity of the M. truncatula trihelix family, we constructed five comparative syntenic diagrams between M. truncatula and the representative species including three dicotyledonous plants (Arabidopsis, soybean, and alfalfa) and two monocotyledonous (rice and maize) (Figure 4). The details are provided in Table S3. MtGT genes displayed syntenic relationships in different degrees with five species; they had the most collinearity relationship with soybean, followed by alfalfa, and had very little collinearity relationship with the maize and rice. A total of 25 and 22 MtGT genes showed syntenic relationships with soybean and alfalfa, respectively. However, only one and two genes had collinearity relationships with rice and maize, respectively. Clearly, the study of MtGT family genes can provide a more valuable gene functional reference for legume crops.

Figure 4

Syntenic analysis of the MtGT genes between M. truncatula and five representative plant species (A. thaliana, G. max, Z. mays, O. sativa and M. sativa).

3.6. Expression Patterns of MtGT Genes in Different Tissues

The tissue-specific expression data of MtGT genes in six tissues by RNA-seq were retrieved from MTGD (http://www.medicagogenome.org/) (Table S4), including blade, bud, nodule, flower, root, and seedpod, which are shown with a heat map in Figure 5a. Except for the expression data of MtGT-11 and MtGT-30, which were not found, the other 36 MtGT genes had different expression levels in six tissues. Among them, some MtGT genes were highly expressed in specific tissues, MtGT-8, MtGT-9, and MtGT-29 were expressed at relatively high levels in roots, flowers, and blades; MtGT12 was specifically expressed in flowers and seedpods; MtGT4 and MtGT35 were expressed at high levels in roots and nodules; and MtGT31 and MtGT32 were only expressed highly in nodules. However, there were several MtGT genes whose expression levels were similar in six tissues, such as Mt-GT1, MtGT-27, MtGT-20, MtGT-15, MtGT-22, and MtGT-23. In addition, the expression levels of MtGT21 and MtGT24 in various tissues were very low (Figure 5a). The expression levels of nine selected MtGT genes were further verified through qRT-PCR in the six tissues (root, stem, blade, flower, bud and seedpod) (Figure 5b; Table S5). The results demonstrated that tissue expression levels of most selected MtGT genes were consistent with the RNA-seq data from MTGD except MtGT4, suggesting that MtGT family members play various roles in specific tissues during the growth and development of M. truncatula.

Figure 5

Tissue-specific expression characteristics of MtGT genes. (a) The expression heatmap of 36 MtGT genes in six tissues (root, blade, bud, flower, seedpod and nodule) from RNA-seq data. (b) The expression patterns of 9 MtGT genes in six tissues (root, stem, blade, flower, bud and seedpod) were validated by real-time qPCR. Expression levels were normalized using MtActin as the internal control and error bars indicated standard deviation among three biological replicates.

3.7. Expression Profiling Analysis of MtGT Genes in Response to Abiotic Stress

Recent studies have indicated that trihelix genes play crucial roles in plants response to abiotic stresses. Based on the M. truncatula Gene Expression Atlas (https://mtgea.noble.org/v3/), we obtained the 33 MtGT gene chips expression data in the blades of 28-day-old seedlings under drought and salt treatments (Table S6). Through the differential expression analysis of these genes, we found that 12 MtGT genes (MtGT-31, MtGT-32, MtGT-19, MtGT-33, MtGT-28, MtGT-24, MtGT-22, MtGT-23, MtGT-10, MtGT-17, MtGT-9, and MtGT-14) were significantly up-regulated and 5 MtGT genes (MtGT-3, MtGT-4, MtGT-6, MtGT-12, and MtGT-13) were significantly down-regulated under drought stress for 96 h (Figure 6a). Other genes (MtGT-20, MtGT-38, MtGT-16, and MtGT-26) were significantly up-regulated for 72 h of drought treatment. Additionally, the salt stress expression analysis of MtGT genes revealed that 13 MtGT genes (MtGT-14, MtGT-36, MtGT-24, MtGT-22, MtGT-23, MtGT-3, MtGT-26, MtGT-10, MtGT-16, MtGT-12, MtGT-13, MtGT-28, and MtGT-29) were significantly up-regulated, and 8 MtGT genes (MtGT-4, MtGT-2, MtGT-35, MtGT-15, MtGT-18, MtGT-5, MtGT-31, and MtGT-32) were significantly down-regulated under 24-h salt stress (Figure 6b). However, MtGT-8 and MtGT-25 were significantly up-regulated under 2 h of salt stress, and then their expression levels decreased. MtGT-6 was significantly up-regulated under 10-h salt stress. Among these genes, many genes could respond to both drought and salt stress. The expression profiles of nine MtGT genes under drought and salt treatments were validated by qRT-PCR (Figure 6; Table S7). The results showed that the expression characteristics of most genes under drought and salt stress treatments were in accordance with the gene chip data. Particularly for MtGT20, MtGT22, and MtGT33, they were dramatically up-regulation by drought and salt treatments at the same time. Interestingly, MtGT-33 was remarkably up-regulated in blades at 0–2 h under drought stress but down-regulated at 2–24 h; MtGT20 and MtGT22 were continuously up-regulated under drought stress within 96 h. However, in salt stress treatment, MtGT-20 and MtGT-22 were remarkably up-regulated in blades at 2–24 h but down-regulated at 24–48 h; MtGT33 was continuously up-regulated within 48 h. They may be involved in the response regulation of abiotic stress.

Figure 6

Expression profiles of the MtGT genes in response to abiotic stresses. A total of 33 MtGT genes chip expression data of 28-day-old seedlings under drought (40–45% soil water content) (a) and salt (200 mM NaCl) treatments (b). The relative expression levels were -log2 transformed and visualized by heat map. The colors vary from blue to red, and circles from small to large represent the scale of the relative expression levels. The expression patterns of nine MtGT genes under drought and salt treatments were validated by qRT-PCR. Expression data were normalized using MtActin as the internal control and error bars indicate standard deviation among three biological replicates.

3.8. Expression Profiling of MtGT Genes in Response to ABA Treatments

Aforementioned results in this study demonstrated that some MtGT genes could be dramatically induced by drought and salt treatments. The phytohormone abscisic acid (ABA), which is considered as the core adversity signal in plants, plays a critical role in response to abiotic stresses such as drought, salinity, and chilling [47,48,49]. Therefore, we further verified the expression profiles of 15 differentially expressed MtGT genes under exogenous ABA treatment by qRT-PCR (Figure 7; Table S8). Most of the genes, particularly MtGT19, MtGT33, and MtGT35, had a strong response to ABA hormones. Their expression levels increased more than ten or even hundreds of times for 48 h of ABA treatment, indicating that these TFs played vital roles in response to the ABA stress signal.

Figure 7

Expression profiles of 15 selected MtGT genes in response to ABA treatment. Expression data were normalized using MtActin as the internal control and error bars indicate standard deviation among three biological replicates. Asterisks indicate the corresponding genes were significantly upregulated or downregulated compared with the control (* p < 0.05, ** p < 0.01, *** p < 0.001; Student’s t-test).

4. Discussion

Recently, a few more trihelix TFs have been characterized which played important roles in multiple processes during plant growth and development, such as trichome development [14], shattering of the mature seed during crop domestication [17], morphogenesis control of floral organs [13], and response to abiotic and biotic stresses [15,18]. In this study, we identified 38 MtGT genes in M. truncatula, which is similar to the number of trihelix genes in Arabidopsis (30) [12], rice (41) [23], and tomato (36) [24]. Additionally, it is close to the average number of trihelix genes on each subgenome of wheat (31) [29]. The 38 MtGT genes were divided into five clades (GT-1, GT-2, SH4, GTγ, and SIP1) by constructing an unrooted phylogenetic tree to analyze and compare with trihelix family members in Arabidopsis. At present, the functions of several trihelix family genes, such as PETAL LOSS (PTL) gene [13], ARABIDOPSIS 6B-INTERACTING PROTEIN1-LIKE1 (ASIL1) and ASIL2 gene [50], have been studied in depth in Arabidopsis. It is helpful to find some MtGT genes which have similar functions as those reported in Arabidopsis through phylogenetic analysis.

We further analyzed the gene structure and conserved motifs of the trihelix family members in M. truncatula, and the result was consistent with the family classification. We found that most MtGT genes in GT-1, GT-2, and SH4 clades contained the trihelix domain (GT domain), whereas all the members of SIP1 and GTγ subfamilies contained MYB DNA-binding domain. It is consistent with the hypothesis that the trihelix domain originated from a MYB-like gene carrying only one repeat [5]. The most members classifying into the same clade shared similar motif compositions and exon/intron, which indicated that the specific conserved motif may play an important role in the function of a particular cluster. Among five clades, most members of GT-1 and GT-2 clades shared motifs 1, 4, 5, and 7, which had higher homology between them than that in other subfamilies of M. truncatula. This is similar to the results in Arabidospsis, and several AtGT genes in the GT1 and GT2 clusters have similar functions. GT-1 and GT-3a of GT1 clade, GT-2 and DF1-like of GT2 clade are involved in light-induced response [5,9,10,11,51]; EMB2746 (GT1) and EDA31 (GT2) were identified as essential for Arabidopsis embryo development [52,53]. As the largest subfamily, the composition of motifs of the SIP1 (most of the members of this clade shared motifs 1, 2, 3, and 4) was quite different from that of other subfamily members, whose composition of motifs was similar to that in cabbage [25], chrysanthemum [28], and wheat [29]. The functions of SIP1 members may be more complex and diverse in the trihelix family of M. truncatula.

Gene duplication including tandem, segmental, and genomic duplication have significant impacts on the generation of novel genes and functional diversity, facilitating the evolution and expansion of gene families in plant genomes [25,45]. There were three groups of MtGT genes with tandem duplication events and two pairs of MtGT genes with segmental duplication in trihelix genes family of M. truncatula. Compared with soybean (67) [27], P. trichocarpa (56) [30], and B. Rapa (52) [25], the number of MtGT genes with duplication events were fewer in M. truncatula. We speculated that most trihelix family genes originate from the different ancestors and are less conservative in M. truncatula. Additionally, these results indicated that gene functions of the M. truncatula trihelix family may have a high degree of divergence and diversity.

The tissue-expression pattern is an important factor in the study of gene functional characteristics. Based on the RNA-seq data combined with qRT-PCR verification, we can hypothesize that MtGT TFs play specific and significant roles in the growth and development of M. truncatula. Most of the MtGT genes presented a tissue-specific expression pattern in M. truncatula. Particularly, MtGT12 was specifically expressed in flowers and seedpods, and MtGT6 from GT-2 clade exhibited relatively high expression in flowers, which may affect the development of M. truncatula floral organs and embryoid. The PETAL LOSS (PTL) gene encoding a GT-2 TF repressed growth in the sepal whorl in Arabidopsis. The ptl mutants exhibited missing petals and partial fusion of sepal whorl [13]. The EDA31, a close member of PTL, was found to be involved in embryo sac development due to defective polar fusion in its mutant [53]. MtGT4 and MtGT35 were expressed at high levels in roots and nodules, and MtGT31 and MtGT32 were only expressed highly in nodules. This is consistent with the nature of M. truncatula as a typical legume with developed root system and strong nitrogen fixation ability. Tissue-specific genes may play vital roles in the growth and differentiation of the corresponding organs or tissues, but further experiments are needed to verify the biological function of these MtGT genes.

Recent studies have reported that trihelix genes are involved in the ABA signalling pathway in response to plant abiotic stresses. The accumulation of ABA causes stomatal closure in guard cells to prevent water loss and regulates the expression of numerous genes to induce various cellular and molecular events, such as second messenger Ca2+ signal system and antioxidant enzyme system, to improve stress tolerance [54,55,56]. Yu et al., reported that ShCIGT belonging to the GT-1 subfamily was involved in the regulation of abiotic stresses resistance in tomato by interacting with a mediator of ABA signal: SNF1-related protein kinase 1 (SnRK1) [57]. Additionally, previous studies have shown that trihelix TFs were involved in Ca2+ signal regulation in response to abiotic stress. AtGT-3b was dramatically induced by salt stress and could interact with the GT-1 cis-element of the SCaM-4 (CaM isoform) promoter in soybean responding to various environmental stresses [18]. Overexpression of AtGT2L which act as a Ca2+-dependent CaM-binding protein involved in plant stress response enhanced the tolerance to cold and salt stress in Arabidospsis [58]. Furthermore, overexpression of Arabidopsis SIP1 clade trihelix 1 (AST1) could regulate the expression of multiple physiological response genes, including proline biosynthesis genes, LEA family genes, POD and SOD genes, improved drought and salt stress tolerance [59]. Based on the aforementioned results, among MtGT family genes, MtGT10, MtGT19, MtGT20, MtGT22, and MtGT33 were significantly induced by drought, salt, and ABA treatments, demonstrating their important roles in abiotic stresses response and resistance in M. truncatula. This study provides a valuable reference gene resource for the molecular genetic improvement of stress resistance in legumes, particularly in alfalfa. The way they participate in the ABA signalling response or interact with the Ca2+ signal pathway, regulatory pathways they are involved in and their role (positive or negative), and functional genes they interact with need to be further investigated.