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Article

Genome-Wide Identification and Characterization of the GASA Gene Family in Medicago truncatula, and Expression Patterns under Abiotic Stress and Hormone Treatments

by
Cai Gao
,
Zhongxing Li
,
Hanwen Zhang
,
Chun Li
,
Haoyang Sun
,
Shuo Li
,
Nan Ma
,
Xiangyu Qi
,
Yilin Cui
,
Peizhi Yang
* and
Tianming Hu
*
College of Grassland Agriculture, Northwest A&F University, Xianyang 712100, China
*
Authors to whom correspondence should be addressed.
Plants 2024, 13(17), 2364; https://doi.org/10.3390/plants13172364 (registering DOI)
Submission received: 31 May 2024 / Revised: 9 August 2024 / Accepted: 21 August 2024 / Published: 24 August 2024
(This article belongs to the Section Plant Genetics, Genomics and Biotechnology)
Browse Figures
Versions Notes

Abstract

:
Medicago truncatula is a key model plant for studying legume plants, particularly alfalfa (Medicago sativa), due to its well-defined genetic background. Plant-specific GASA (Gibberellic Acid Stimulated Arabidopsis) genes play various roles in plant growth and development, abiotic stress, and hormone responses. However, limited information is available on GASA research in Medicago. In this study, 26 MtGASAs were identified and analyzed for its structure, evolution, and expressions. Sequence alignments and phylogeny revealed that 26 MtGASAs containing conserved GASA domains were classified into three clades. The chromosomal locations and gene synteny revealed segmental and tandem repetition evolution. Analysis of cis-regulatory elements indicates that family members likely influence various hormone signaling pathways and stress-related mechanisms. Moreover, the RNA-seq and qRT-PCR analyses revealed that 26 MtGASAs were extensively involved in abiotic stresses and hormone responses. Notably, seven MtGASA genes (MtGASA1, 10, 12, 17, 23, 25 and 26) were all dramatically activated by NaCl and Mannitol treatments, and four MtGASAs (MtGASA7, 10, 23 and 24) were significant activated by GA3, PBZ, ABA, and MeJA treatments. Collectively, this study is the first to identify and describe GASA genes in Medicago on a genome-wide scale. The results establish a basis for functional characterization, showing that these proteins are essential in responding to various abiotic stresses and hormonal signals.

1. Introduction

Gibberellic Acid Stimulated Arabidopsis (GASA) is a cysteine-rich peptide (CRP) protein and a low-molecular-weight peptide found widely in plants [1] (Bouteraa et al., 2023). It is activated by gibberellin (GA), which influences plant growth and development [2,3,4,5,6] (Herzog et al., 1995; Almasia et al., 2008; Zimmermann et al., 2010; Nahirñak et al., 2012; Wu et al., 2021). Members of the GASA family exhibit a conservative protein structure, featuring a signal peptide at the N-terminal, a variable region, and a cysteine domain at the C-terminal [7] (Silverstein et al., 2007). The cysteine domain typically contains 12 cysteine residues and plays a crucial role in the function of GASA family proteins [8] (Sun et al., 2013).
The first GASA gene, GA-stimulated transcript 1 (GAST1), was identified in tomato and known as the downstream gene responding to gibberellin acid [9] (Shi et al., 1992). The GASA family genes have been identified in various species including Malus domestica [10] (Fan et al., 2017), Glycine max [11] (Ahmad et al., 2019), Oryza sativa [12] (Muhammad et al., 2019), Vitis vinifera [13] (Ahmad et al., 2020), Gossypium hirsutum [14] (Qiao et al., 2021), Populus trichocarpa [15] (Wu et al., 2022a), Arachis hypogaea [16] (Wu et al., 2022b), Nicotiana tabacum [17] (Li et al., 2022b), Citrus clementina [6] (Wu et al., 2021), and Solanum lycopersicum [18] (Su et al., 2023).
GASA family genes have been reported to be involved in abiotic stresses, hormone signals, and plant development. GsGASA1 is involved in inhibiting root growth induced by cold [19] (Li et al., 2011). AhGASA6, 23, and 5, which are homologous genes of AtGASA1 and AtGASA11, respectively, have been reported to be related to peanut seed and shell development [16] (Wu et al., 2022b). AtGASA14 is positively regulated by GA and plays a role in both abiotic stress resistance and leaf expansion [8] (Sun et al., 2013). SmGASA4, a gene from Salvia miltiorrhiza, enhances flower and root development in Arabidopsis by positively regulating gibberellin, boosting plant resistance to salt, drought, and paclobutrazol (PBZ, a gibberellin synthesis inhibitor) stress [20] (Wang et al., 2018). GhGASA10-1 has been shown to enhance cotton fiber elongation by regulating IAA-induced cellulose synthesis [21] (Chen et al., 2021). AtGASA5 suppresses heat stress by mediating the interaction between gibberellin and salicylic acid (SA) signaling [22] (Zhang and Wang, 2011). AtGASA4 and AtGASA6 are upregulated by GA and downregulated by stress hormones like ABA and jasmonic acid (JA) [23] (Qu et al., 2016). Furthermore, AtGASA4, AtGASA5, and AtGASA6 have been implicated in plant flowering [23,24,25] (Roxrud et al., 2007; Zhang et al., 2009; Qu et al., 2016). GEG (Gerbera hybrida homolog of the gibberellin [GA]–stimulated transcript 1 [GAST1] from tomato), a GASA family member in Gerbera hybrida, is directly activated by GhMIF protein to regulate petal elongation [26] (Han et al., 2017).
Medicago truncatula has become an ideal model plant for studying legumes due to its low ploidy, small genome size, and high genetic transformation efficiency [27] (Cui et al., 2022). Legumes are essential in human diet and livestock feed due to their high-quality plant protein content. The growth, development, and resistance of forage grass are closely linked to achieving high yield and quality. However, the information on GASA genes and their function in Medicago truncatula are still unclear. Therefore, identifying and characterizing GASA genes is essential for M. truncatula breeding to withstand challenging environmental conditions and promote improved growth.
In this study, 26 GASA genes in M. truncatula were identified. We analyzed characteristics of 26 family members, including gene length, structure, molecular weight, protein features, and phylogenetic traits. Additionally, we systematically studied their chromosome localization and gene duplication events. It was also discovered that MtGASA genes exhibit spatial expression profiles and react to environmental stress. This study offers valuable insights for further exploring the functional characteristics of MtGASAs in Medicago and their potential application in enhancing genetic traits of legume plants to combat abiotic stresses and respond to hormones.

2. Materials and Methods

2.1. Plant Materials, Growth Condition and Treatments

The M. truncatula ecotype R108 was used in this study. To ensure the germination uniformly, seeds were sandpapered to break the hard seed coat and put on wet paper for 48 h in the dark at 4 °C, then moved to 24 °C to wait for sprouting. Seedlings were moved to a 1/2 Hoagland solution and placed in a growth chamber at 24 ± 2 °C under long-day conditions (16/8 h day/night) with 60% humidity (PLT-BRS-15PF, Ningbo Prandt Instrument Co., Ltd., Ningbo, China).
For treatments, the three-week-old plants were grown hydroponically in 1/2 Hoagland supplemented with 200 mM NaCl, 300 mM Mannitol, 100 μM GA3, 50 μM PBZ, 100 μM ABA, and 50 μM methyl jasmonate (MeJA), separately [28,29,30,31,32] (Shu et al., 2016; Yang et al., 2021; Li et al., 2022a; Zhu et al., 2021; Wang et al., 2022). The leaves were harvested at 0, 1, 3, 6, and 12 h after treatment for subsequent analysis. Three biological replicates were used in each experiment.

2.2. Identification of GASA Family Member in M. truncatula

Fifteen GASA protein sequences from A. thaliana have been reported [24,33] (Roxrud et al., 2007; Zhang and Wang, 2008), and these were obtained from The Arabidopsis Information Resource (TAIR) database. These sequences were then used in a BLASTP search against the M. truncatula A17 protein dataset (https://medicago.toulouse.inra.fr/MtrunA17r5.0-ANR/; accessed on 10 May 2023) [34] (Pecrix et al., 2018) with an E-value ≤ 10−7 parameter. Then, the GASA structure domain file (Pfam: PF02704) and HMMER v3.3.2 software [35] (Finn et al., 2015) were used to search for MtGASA genes. To determine whether the candidate GASA sequences contained the conserved domain, the NCBI-CDD [36] (Wang et al., 2023) and SMARTdatabases [37] (Letunic et al., 2021) were used. In addition, the Expasy ProtParam tool [38] (Gasteiger et al., 2003) was utilized to determine characteristics including molecular weight, theoretical pI (isoelectric point), amino acid count, instability index, and grand average of hydropathicity (GRAVY).

2.3. Chromosomal Localization and Gene Duplication

The M. truncatula A17 genomic database (https://medicago.toulouse.inra.fr/MtrunA17r5.0-ANR/) was used to retrieve the gene annotations and chromosomal locations of 26 MtGASAs. The gene locations on chromosomes were accurately plotted using Mapchart 2.32 software [39] (Voorrips, 2002). The conserved domain in GASA protein sequences and the exon-intron structures were identified and generated using TBtools [40] (Chen et al., 2020). The sequences of GASA proteins in Medicago are shown in Table S2.

2.4. Sequence Alignment and Polygenetic Analysis

MUSCLE (https://www.ebi.ac.uk/Tools/msa/muscle; accessed on 20 June 2023) was used to perform the multiple alignments of GASA protein sequences in M. truncatula. The phylogenetic tree was constructed using the neighbor-joining (NJ) method in MEGA X [41] (Kumar et al., 2018) with 1000 bootstrap replications.

2.5. Tandem Duplication and Synteny Analysis

The Plant Genome Duplication Database [42] (Lee et al., 2013) was used to design the syntenic blocks. Diagrams were constructed using Circos version 0.63 [43] (Krzywinski et al., 2009). The chromosomal location helps determine the tandem duplication of GASA genes in Medicago. Genes with a neighboring homologous GASA gene on the same chromosome, separated by no more than one intervening gene, were classified as tandem duplicates.

2.6. Cis-Regulatory Element Analysis

Promoter regions provide essential insights for predicting gene functions. To find the putative functions in abiotic stress and plant hormones, the 3000 bp upstream sequence of the start codon was chosen from the M. truncatula A17 genome database (https://medicago.toulouse.inra.fr/MtrunA17r5.0-ANR/) as the promoter region and utilized for cis-regulatory element analysis through the PlantCARE Database [44] (Lescot et al., 2002).

2.7. MtGASA Gene Expression Profiles under Multiple Tissues and Abiotic Stresses

RNA-seq data from six different tissues (root, bud, nodule, seedpod, leaf, and flower; Accession Number: SRX099057–SRX099062) [45] (Young et al., 2011) and abiotic stresses (whole seedlings samples) (cold, freeze, salt, drought, and ABA; Accession Numbers: SRX1056987–SRX1056992) [28] (Shu et al., 2016) in M. truncatula were downloaded from the NCBI SRA database (Table S3) and cleaned using fastp v0.20.0 [46] (Chen et al., 2018). The clean reads were then mapped to the M. truncatula A17 genome sequence using HISAT2 v2.2.0 software [47] (Pertea et al., 2016). The FPKM values were calculated using Cufflinks v2.2.1 software [48] (Trapnell et al., 2012), representing fragments per kilobase of transcript per million mapped fragments.

2.8. Quantitative RT-PCR Analysis

Eastep® Super Total RNA Extraction Kit (Promega, Shanghai, China) was chosen to extract the total RNA from samples. Subsequently, cDNA was synthesized from each sample by utilizing 1 µg of total RNA according to the instructions of the HiScript III RT SuperMix for qPCR (+gDNA wiper; Vazyme Bio, Nanjing, China). All the primers were designed for real-time PCR on the website IDT (https://sg.idtdna.com/scitools/Applications/RealTimePCR/; accessed on 3 August 2023) and checked for specificity on the NCBI website. Real-time PCR was carried out in a Real-Time PCR Detection System (Roche). The reaction used ChamQ SYBR qPCR Master Mix (Vazyme Bio, Nanjing, China), following the thermal cycling conditions described in previous studies [49] (Yang et al., 2023). The gene MtActin was utilized as an internal control to standardize all mRNA expression levels [50] (Zhang et al., 2016). The amount of template in each PCR amplification mixture was assessed using the 2−∆∆Ct method, with mean values derived from three independent PCR amplifications. The detailed primer information is shown in Table S4.

3. Results

3.1. Identification and Annotation of GASA Genes in Medicago truncatula

In order to identify all GASA proteins in M. truncatula and investigate their function, we searched for the conserved GASA domain (PF02704) by conducting a multi-sequence alignment with the protein sequences of AtGASAs in the whole genome protein database. Sequences with high similarity (E-value lower than 10−5) were retrieved from the database. After removing incomplete and redundant sequences, a total of 26 GASAs were identified in the protein database.
The 26 MtGASAs were renamed sequentially according to their location and order on the chromosomes. The protein length of these genes ranged from 65 to 219 amino acids, showing a wide distribution of gene length (Table S1). The MtGASA proteins ranged in molecular weight from 7.39 to 23.65 kDa, with isoelectric point (pI) values between 5.75 and 9.65 (Table S1). However, over half of the pI values were higher than 7, which indicates that most family members are alkaline proteins. Furthermore, only 7 GASA proteins had an Instability index less than 40, meaning they are stable proteins.

3.2. Systematic Phylogeny, Gene Structure, and Motif Analysis of GASA Genes in M. truncatula

To investigate the phylogeny among GASA genes, protein sequences (26 MtGASAs, 15 AtGASAs, 10 OsGASAs, and 40 AhGASAs) were used to construct a phylogenetic tree using the NJ method, which showed that GASAs were clustered into three groups (Groups 1, 2, and 3) (Figure 1). Groups 1, 2, and 3 have 13, 3, and 10 MtGASAs, respectively. GASA genes in the same group have a close relationship, which might be derived from a common ancestral gene. Moreover, the sequence similarity of M. truncatula-A. thaliana (Figure S1) and M. truncatula-M. truncatula (Figure S2) GASA proteins illustrated the same conclusion.
Diversity in exon-intron structures is crucial for the evolution of gene families and provides additional evidence to support phylogenetic classifications (Figure 2A). More than half of MtGASA genes possessed 2 to 4 introns, while five MtGASAs (MtGASA1, 5, 9, 16, and 20) had one intron, and five MtGASAs (MtGASA6, 11, 14, 15, and 18) had no intron (Figure 2B). Motif analysis can explore protein structure diversity and predict potential functions. Ten conserved motifs were found among the family members (Figure 2C). Among those, motif 1 was present in all MtGASAs and stood out as the most conserved. Motif 2 was predominantly located at the C terminus, with motif 3 found at the N terminus. The findings suggest that closely related members share a similar motif arrangement, whereas motifs vary significantly among different groups or subgroups. All 26 MtGASAs contain three conserved motifs: motifs 1 to 3. Motifs 1 and 2 constitute the GASA domain, whereas motif 3 is associated with the conserved signal peptide of these family. All predicted GASA genes containing conserved motifs were highly invariable. The results in Figure 2 indicated that the gene structure and motif distribution show consistent affinities with the phylogenetic tree.
In previous research, GASA proteins have been shown to contain a C-terminal domain with 12 conserved cysteines that are highly conserved [51] (Aubert et al., 1998), which control all biological activities of the GASA proteins [10,13] (Ahmad et al., 2020; Fan et al., 2017). The results in Figure 3 reveal that 26 MtGASA proteins share the conserved domain as reported, apart from MtGASA11 because of its mutated GASA domain.

3.3. Chromosomal Localization and Gene Duplication Analysis of MtGASAs

The 26 MtGASA genes were located on seven chromosomes in M. truncatula, except Chromosome (Chr) 2 (Figure 4A). Each chromosome contained 2 to 6 MtGASAs. In particular, Chr 5 contained a maximum number of 6 MtGASAs, followed by Chr 1 and Chr 3 with 5 MtGASAs on each. In addition, there were 4 MtGASAs on Chr 6, while Chr4, 7, and 8 contained 2 MtGASAs, respectively. Such a prejudiced distribution pattern of GASA genes also occurs in the apple genome [10] (Fan et al., 2017).
Gene clusters, or tandem duplication on chromosomes, form ‘hot spots’. Several substantial clustering or tandem duplications were found in the MtGASA gene family (Figure 4B). For example, Chr 5 contained 4 MtGASAs (MtGASA13–16) that are segmental duplications in a local region. Moreover, 3 MtGASAs (MtGASA19, 20, and 21) are considered tandem duplications on Chr 6, which are homologs of OsGASA3 on chromosome 4.

3.4. Promoter Region Analysis of MtGASAs

To find out the potential biological functions of MtGASAs, promoter regions of 26 MtGASA genes were analyzed through the PlantCARE Database. Cis-acting elements have been suggested to be involved in plant growth and development, as well as responses to abiotic stresses, hormones, and light (Figure 5A). Specifically, 21 out of 26 MtGASA promoters were found to contain AAGAA motifs, which are cis-acting regulatory elements associated with plant growth and development. Except MtGASA20, the other MtGASA promoters have at least one G-box or box 4 elements, indicating these genes exhibit a close correlation with the processes of light responsiveness (Figure 5B). A total of 93 MYC and 108 MYB binding elements associated with abiotic stress response were identified in the promoters of MtGASAs. Notably, the MtGASA10 promoter contains 30 abiotic stress-associated elements, making it the most numerous of all MtGASAs promoters. (Figure 5B). MtGASA7, 12, 17, 20, and 24 contain one to three of the gibberellin response elements (P-box, GARE- and TATC-motif) in its promoter region. In addition, there are the cis-acting elements that play a role in the responsiveness to various hormones. These include the CGTCA-motif and TGACG-motif for MeJA signaling, TCA-element for SA reactivity, and ABRE for ABA sensitivity. Overall, MtGASAs are possibly regulated through binding of their cis-regulatory elements.

3.5. Expression Patterns of MtGASAs

To obtain the expression pattern of MtGASAs, we performed an RNA-seq analysis using abiotic treatments and tissue-specific expression data. As shown in Figure S3A, there were 7 MtGASAs (MtGASA1, 2, 3, 4, 10, 12, and 24) positively regulated by ABA treatment, and 7 MtGASAs (MtGASA5, 9, 17, 19, 22, 25, and 26) responded to the cold and freezing treatments. Notably, MtGASA22 and MtGASA23 were specifically expressed in salt stress. Furthermore, tissue-specific expression analysis (Figure S3B) revealed that there were 10 MtGASAs (MtGASA1, 2, 3, 4, 7, 8, 20, 21, 24, and 26) highly expressed in open flowers, 8 MtGASAs (MtGASA12, 13, 14, 15, 16, 19, 23, and 25) were upregulated in the seedpod, and 3 MtGASAs (MtGASA5, 10, and 22) specifically expressed in 4-week buds. Interestingly, only MtGASA9 was specifically expressed in the nodule, and MtGASA17 was unusually expressed in 4-week roots.
Due to the gene similarity and duplication, 12 representatives MtGASAs were further validated through qRT-PCR analysis in combination with promoter elements analysis and RNA-seq data. The levels of expression for these selected genes were analyzed in six tissues (Figure 6): root, stem, leaf (4-week), flower, leaf (8-week), and pod (15-day-old). Seven MtGASAs (MtGASA1, 2, 3, 12, 24, 25, and 26) exhibited high transcript levels in the flower, and MtGASA9 was preferentially expressed in the pod, but MtGASA17 was specifically expressed in the root, aligning with the RNA-seq data. Notably, the expression level of four genes (MtGASA7, 10, 12, and 24) have dramatically huge differences in the leaf between 4 weeks and 8 weeks, suggesting that they may play a core role in growth and development.
To exploring the expression levels of the selected 12 MtGASAs in various conditions, we examined two abiotic stresses (NaCl and Mannitol), as well as three hormones (GA3, MeJA, ABA), along with PBZ treatments. In general, the majority of MtGASA genes were significantly regulated by the various treatments. When exposed to NaCl and Mannitol stresses (Figure 7), five MtGASAs (MtGASA2, 3, 7, 9, and 24) were obviously downregulated during the time, while seven MtGASAs (MtGASA1, 10, 12, 17, 23, 25, and 26) were notably upregulated to a significant extent.
In the GA3 treatment (Figure 8A), the expression levels of seven MtGASAs (MtGASA7, 10, 12, 17, 23, 25, and 26) were greatly activated, as opposed to the genes (MtGASA2, 3, 9, and 24) that were significantly suppressed by GA3. However, MtGASA1 did not exhibit a significant increase in response to GA3 supplementation. For PBZ treatment (Figure 8B), the expression levels of five MtGASAs (MtGASA1, 7, 9, 23, and 26) were obviously promoted. Interestingly, three other MtGASAs (MtGASA2, 3, and 12) were also markedly upregulated but had a peak at 6 h, while the expression level of MtGASA24 significantly decreased within the first 6 h but increased sharply at 12 h. However, MtGASA10, 17, and 25 had no remarkable increase during the treatment. Under ABA treatment (Figure 8C), seven MtGASAs (MtGASA1, 9, 10, 17, 23, 25, and 26) exhibited a notable increase in their expression levels. In contrast, only MtGASA24 was remarkably diminished after 1 h ABA treatment. Additionally, MtGASA7 was only effectively upregulated after 1 h, and the other three genes (MtGASA2, 3, and 12) were upregulated after 6 h. Regarding MeJA (Figure 8D), 10 MtGASAs (MtGASA1, 7, 9, 10, 12, 17, 23, 24, 25, and 26) were evidently increased. Notably, four (MtGASA7, 10, 23, and 24) were sharply activated at 12 h, and the others peaked at 3 h and 6 h, respectively. Interestingly, MtGASA2 and MtGASA3 had the same tendency in that they were activated at 6 h but remarkably diminished at 12 h.

4. Discussion

Gibberellin is a crucial hormone for plant growth and development, playing key roles in seed germination, internode elongation, and flowering. It interacts with other hormones and stresses through a complex network. Numerous genes are involved in the gibberellin pathway response. GASAs are important genes activated by gibberellic acid that regulate target genes or interact with partners to influence various processes. In this study, 26 MtGASAs were found in the M. truncatula genome while searching for GASA genes.
Phylogenetic analysis revealed that MtGASA7, 17, 23, and 24 were clustered with three AtGASAs (AtGASA4, 5, and 6) in Group 1 (Figure 1), which plays a vital role in regulating Arabidopsis flowering [23,25] (Zhang et al., 2009; Qu et al., 2016). The expression levels of MtGASA7, 23, and 24 in young leaves (4-week) were significantly higher than those in old leaves (8-week) (Figure 6), indicating that they may have similar functions in flowering. SlGASA1, the AtGASA1 homologous gene in tomato, was discovered to inhibit the activation of ACS2 and ACO1, resulting to participate in fruit ripening [18] (Su et al., 2023). AhGASA6 and AhGASA23 in peanut, the homologous genes of AtGASA1, have been reported to be related to pod development [16] (Wu et al., 2022b). MtGASA2, 3, and 10 were clustered with AtGASA1, and all the three MtGASAs were regulated by GA3, ABA, and MeJA (Figure 8). In addition, MtGASA10 was highly expressed in the seedpod (Figure 6). Therefore, MtGASA10 may promoted the seedpod development, and the three genes (MtGASA2, 3 and 10) may be involved in interactions between gibberellin and other hormones [52,53] (Nahirnak et al., 2019; Almasia et al., 2020).
Conserved domain analysis elucidated that MtGASA family members mainly contain all 10 motifs (Figure 2), and there were some differences among the groups. Multi-sequence alignments showed high identity among all 26 MtGASA protein sequences in the cysteine domain region (Figure 3). Therefore, all 26 MtGASAs display a highly conserved protein feature throughout evolution. It is important to further understand how plants regulate these members to modulate specific physiological processes. Promoter analysis revealed the elements contained in MtGASA family members and suggested the candidate regulatory signals for these genes (Figure 5).
To gain a better understanding of the biological functions of MtGASAs, we conducted a comprehensive analysis by integrating gene expression, phylogenetic analysis, and synteny analysis. For example, MtGASA12 was specially up regulated by ABA treatment, with minimal expression levels in other stresses, but its ortholog gene MtGASA25 exhibited the highest expression levels under cold stress (Figure S3). Both were upregulated by GA3 in qRT-PCR analysis (Figure 8A), but interestingly, their closest ortholog gene, AtGASA1 in Arabidopsis, is repressed by GA3 [33] (Zhang and Wang, 2008), indicating that functional characteristics may vary among different species. MtGASA7 and MtGASA23 exhibit a strong correlation with AtGASA4 (Figure 1); the notable difference in MtGASA7 expression levels between young and old leaves (Figure 6) indicates a potential role in controlling the flowering process [23] (Qu et al., 2016).
Improving salt and drought tolerance are key objectives for enhancing Medicago. Based on the qRT-PCR analyses under NaCl and Mannitol treatments, seven MtGASAs (MtGASA1, 10, 12, 17, 23, 25, and 26) were highly induced, while the other five genes (MtGASA2, 3, 7, 9, and 24) exhibited an opposing expression pattern (Figure 7). Genes with the closest phylogenetic relationship to the selected members have been reported to have the similar functions. For instance, MtGASA9 responded to mannitol and NaCl stresses, similar to the function of AtGASA14 and OsGASA1 in abiotic stress resistance [8,12] (Sun et al., 2013; Muhammad et al., 2019).
In this study, we discovered that nearly all 12 MtGASAs were regulated by GA3, PBZ, ABA, and MeJA phytohormones (Figure 8). MtGASA2, 3, 9, and 24 have an extremely contrary expression level in GA3 and PBZ treatment, which suggests that these genes vary with the level of GA3 in plants. The effects of ABA and JA on plants in promoting leaf senescence and stomatal closure are consistent with each other [54] (An et al., 2022). Five MtGASAs (MtGASA2, 3, 10, 12, and 26) were induced by ABA treatment, and a similar expression pattern also appeared in MeJA treatment, indicating that the five MtGASAs are probably involved in the crosstalk between ABA and JA signals. AtGASA4 and AtGASA6 are activated by GA but suppressed by ABA and JA [23] (Qu et al., 2016). Similarly, MtGASA7 exhibits a comparable expression profile when treated with GA and ABA. In contrast, MtGASA17 and MtGASA23 demonstrate unique expression patterns compared to MtGASA7 (Figure 1 and Figure 8). These findings indicate that different GASA genes have diverse functions in response to various treatments.
Overall, GASA is a crucial gene in plants that plays a key role in regulating plant development and stress responses. Its complex regulatory network suggests a potential for manipulation of this gene to boost the plant’s ability to resist stress, indicating the potential of this gene as a useful biotechnological tool with a wide range of applications in crop improvement.

5. Conclusions

This study thoroughly investigated cysteine-rich peptides in M. truncatula, characterizing 26 full-length GASA proteins classified into three main clusters based on phylogenetic relationships. Analyzing synteny and comparing GASA genes from various plant species offers valuable insights into the evolutionary roles of MtGASA genes. Additionally, the analysis of MtGASAs expression profiles in various tissues and under different treatments using RNA-seq data and qRT-PCR validation highlighted their significant role Medicago growth, development, and stress tolerance. Specifically, MtGASA7, MtGASA10, MtGASA17, and MtGASA24 were highlighted. This study provides new insights into stress-resistant and hormone-responsive MtGASAs by analyzing phylogenetic relationships, gene structure, and expression patterns. It establishes a theoretical foundation for understanding the specific roles of GASA genes in Medicago.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/plants13172364/s1, Figure S1. The similarity of GASAs between M. truncatula and A. thaliana; Figure S2. The similarity of GASAs between M. truncatula itself; Figure S3. Expression analysis of MtGASA genes under different treatments and various tissues; Table S1. The detailed information of MtGASAs; Table S2. The protein sequences of GASAs in M. truncatula, A. thaliana, O. sativa and A. hypogaea; Table S3. The FPKM of MtGASAs in various tissues and different treatments; Table S4. The primers used in this study.

Author Contributions

Conceptualization, C.G., P.Y. and T.H.; Formal analysis, H.Z., N.M., X.Q. and Y.C.; Investigation, C.G.; Methodology, C.G., C.L., H.S. and S.L.; Project administration, P.Y. and T.H.; Software, Z.L.; Visualization, C.G., Z.L., H.Z., C.L., H.S. and S.L.; Writing—original draft, C.G.; Writing—review and editing, C.G., Z.L., P.Y. and T.H. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the China Agriculture Research System of MOF and MARA, the National Natural Science Foundation of China (31772660), the Key Research and Development Program of Shaanxi Province (2019ZDLNY05-01) and Shaanxi Postdoctoral Research Project (2023BSHEDZZ123).

Data Availability Statement

Data are contained within the article and Supplementary Materials.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Phylogenetic tree of GASA genes of Medicago truncatula, Oryza sativa, Arachis hypogaea, and Arabidopsis thaliana. Blue-colored stars represent M. truncatula proteins, red-colored squares represent A. thaliana proteins, black-colored circles represent O. sativa proteins, and gray-colored triangles represent A. hypogaea proteins. G1, G2, and G3 indicates Group 1, Group 2, and Group 3, respectively, highlighted by different colored oval shapes.
Figure 1. Phylogenetic tree of GASA genes of Medicago truncatula, Oryza sativa, Arachis hypogaea, and Arabidopsis thaliana. Blue-colored stars represent M. truncatula proteins, red-colored squares represent A. thaliana proteins, black-colored circles represent O. sativa proteins, and gray-colored triangles represent A. hypogaea proteins. G1, G2, and G3 indicates Group 1, Group 2, and Group 3, respectively, highlighted by different colored oval shapes.
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Figure 2. Phylogenetic relationships, gene structure, and architecture of conserved motifs in MtGASA genes. (A) Phylogenetic tree of M. truncatula GASA genes. (B) Exon-intron distribution. The purple and gray boxes represent the CDS and UTR region, respectively. CDS denotes exons, and the short black lines represent introns. (C) Motif analysis. The different colors of boxes denote different motif numbers. The length of box indicates motif length.
Figure 2. Phylogenetic relationships, gene structure, and architecture of conserved motifs in MtGASA genes. (A) Phylogenetic tree of M. truncatula GASA genes. (B) Exon-intron distribution. The purple and gray boxes represent the CDS and UTR region, respectively. CDS denotes exons, and the short black lines represent introns. (C) Motif analysis. The different colors of boxes denote different motif numbers. The length of box indicates motif length.
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Figure 3. Alignment of the GASA domain from MtGASA proteins. The red lines represent their conserved cysteines.
Figure 3. Alignment of the GASA domain from MtGASA proteins. The red lines represent their conserved cysteines.
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Figure 4. Chromosomal distribution and synteny analysis of GASA genes in Medicago. (A) The chromosomal location of 26 MtGASAs. The different colored lines indicated the different syntenic gene pairs. (B) The examples of fragment duplication and tandem duplication during evolution. The arrows indicate the position and orientation of genes on chromosomes.
Figure 4. Chromosomal distribution and synteny analysis of GASA genes in Medicago. (A) The chromosomal location of 26 MtGASAs. The different colored lines indicated the different syntenic gene pairs. (B) The examples of fragment duplication and tandem duplication during evolution. The arrows indicate the position and orientation of genes on chromosomes.
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Figure 5. Cis-regulatory element prediction in the MtGASA promoters. (A) The total number of each type of cis-regulatory element and element types are marked by different colors. (B) Detailed information of the cis-regulatory elements in MtGASA promoters. Colors and numbers of the grid indicate the numbers of different cis-regulatory elements in MtGASA genes.
Figure 5. Cis-regulatory element prediction in the MtGASA promoters. (A) The total number of each type of cis-regulatory element and element types are marked by different colors. (B) Detailed information of the cis-regulatory elements in MtGASA promoters. Colors and numbers of the grid indicate the numbers of different cis-regulatory elements in MtGASA genes.
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Figure 6. Expression analysis of 12 MtGASA genes in six tissues by qRT-PCR analysis. Data are normalized to the actin gene (one-way ANOVA was performed, and statistically significant differences are indicated by lettered labels).
Figure 6. Expression analysis of 12 MtGASA genes in six tissues by qRT-PCR analysis. Data are normalized to the actin gene (one-way ANOVA was performed, and statistically significant differences are indicated by lettered labels).
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Figure 7. Expression profiles of 12 representative MtGASA genes under abiotic stress treatments. (A) Mannitol treatment. (B) NaCl treatment. (* p < 0.05, ** p < 0.01, *** p < 0.001, Student’s t-test).
Figure 7. Expression profiles of 12 representative MtGASA genes under abiotic stress treatments. (A) Mannitol treatment. (B) NaCl treatment. (* p < 0.05, ** p < 0.01, *** p < 0.001, Student’s t-test).
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Figure 8. Expression profiles of 12 representative MtGASA genes in response to different hormonal treatments. (A) GA3 treatment. (B) PBZ treatment. (C) ABA treatment. (D) MeJA treatment. (* p < 0.05, ** p < 0.01, *** p < 0.001, Student’s t-test).
Figure 8. Expression profiles of 12 representative MtGASA genes in response to different hormonal treatments. (A) GA3 treatment. (B) PBZ treatment. (C) ABA treatment. (D) MeJA treatment. (* p < 0.05, ** p < 0.01, *** p < 0.001, Student’s t-test).
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Gao, C.; Li, Z.; Zhang, H.; Li, C.; Sun, H.; Li, S.; Ma, N.; Qi, X.; Cui, Y.; Yang, P.; et al. Genome-Wide Identification and Characterization of the GASA Gene Family in Medicago truncatula, and Expression Patterns under Abiotic Stress and Hormone Treatments. Plants 2024, 13, 2364. https://doi.org/10.3390/plants13172364

AMA Style

Gao C, Li Z, Zhang H, Li C, Sun H, Li S, Ma N, Qi X, Cui Y, Yang P, et al. Genome-Wide Identification and Characterization of the GASA Gene Family in Medicago truncatula, and Expression Patterns under Abiotic Stress and Hormone Treatments. Plants. 2024; 13(17):2364. https://doi.org/10.3390/plants13172364

Chicago/Turabian Style

Gao, Cai, Zhongxing Li, Hanwen Zhang, Chun Li, Haoyang Sun, Shuo Li, Nan Ma, Xiangyu Qi, Yilin Cui, Peizhi Yang, and et al. 2024. "Genome-Wide Identification and Characterization of the GASA Gene Family in Medicago truncatula, and Expression Patterns under Abiotic Stress and Hormone Treatments" Plants 13, no. 17: 2364. https://doi.org/10.3390/plants13172364

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