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Article

Genome-Wide Analysis of the MADS-box Gene Family and Expression Analysis during Anther Development in Salvia miltiorrhiza

by
Songyue Chai
1,2,†,
Kexin Li
1,2,†,
Xuexue Deng
1,2,
Long Wang
1,2,
Yuanyuan Jiang
1,2,
Jinqiu Liao
1,3,
Ruiwu Yang
1,3 and
Li Zhang
1,2,*
1
Featured Medicinal Plants Sharing and Service Platform of Sichuan Province, Sichuan Agricultural University, Ya’an 625014, China
2
College of Science, Sichuan Agricultural University, Ya’an 625014, China
3
College of Life Sciences, Sichuan Agricultural University, Ya’an 625014, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Int. J. Mol. Sci. 2023, 24(13), 10937; https://doi.org/10.3390/ijms241310937
Submission received: 4 May 2023 / Revised: 26 June 2023 / Accepted: 27 June 2023 / Published: 30 June 2023
(This article belongs to the Special Issue Horticultural Crop Improvement: A New Era for Plant Molecular Research 2.0)
Browse Figures
Versions Notes

Abstract

:
MADS-box genes constitute a large family of transcription factors that play important roles in plant growth and development. However, our understanding of MADS-box genes involved in anther development and male sterility in Salvia miltiorrhiza is still limited. In this study, 63 MADS-box genes were identified from the genome of the male sterility ecotype Sichuan S. miltiorrhiza (S. miltiorrhiza_SC) unevenly distributed among eight chromosomes. Phylogenetic analysis classified them into two types and 17 subfamilies. They contained 1 to 12 exons and 10 conserved motifs. Evolution analysis showed that segmental duplication was the main force for the expansion of the SmMADS gene family, and duplication gene pairs were under purifying selection. Cis-acting elements analysis demonstrated that the promoter of SmMADS genes contain numerous elements associated with plant growth and development, plant hormones, and stress response. RNA-seq showed that the expression levels of B-class and C-class SmMADS genes were highly expressed during anther development, with SmMADS11 likely playing an important role in regulating anther development and male fertility in S. miltiorrhiza_SC. Overall, this study provides a comprehensive analysis of the MADS-box gene family in S. miltiorrhiza, shedding light on their potential role in anther development and male sterility.

1. Introduction

MADS-box (Mini chromosome maintenance 1 (MCM1), Agamous (AG), Deficiens (DEF), and Serum response factor (SRF)) transcription factors constitute one of the largest families in plants [1]. They can be classified into two categories, type I and type II [2]. Type I MADS-box genes typically contain one to two exons and encode proteins with the highly conserved MADS-box (MADS) domain [3], but their biological functions remain largely unknown. In contrast, type II MADS-box genes contain six to eight exons [4] and have four typical domains: MADS, Intervening (I), Keratin-like (K), and C-terminal (C) domains [4,5]. The MADS domain enables DNA binding activity [6], while the I and K domains facilitate the formation of dimers and higher-order complexes between two or more MADS-box proteins [7,8,9], and the C domain contributes to transcriptional activation [6]. Due to this characteristic domain structure, type II genes are also known as MIKC-type MADS-box genes [5]. The MADS-box gene family has been identified and characterized in various plants, such as Arabidopsis thaliana (108) [10], Oryza sativa (75) [11], Zea mays (75) [12], Vitis vinifera (83) [13], and Medicago sativa (120) [14]. However, studies on MADS-box gene families in medicinal plants are limited.
MADS-box genes are crucial regulators of floral organ development [15], which encompass sepals, petals, stamens, and carpels [16]. The genetic control of floral organ development was elucidated by the ABC model, subsequently expanded by the ABCDE model [17]. Using A. thaliana as an example, sepal development is regulated by A-class (APETALA1 (AP1)) and E-class (SEPALLATA (SEP)) genes, while petal development is regulated by A-class, B-class (AP3 and PISTILLATA (PI)), and E-class genes. The formation of stamens is controlled by B-class, C-class (AGAMOUS (AG)), and E-class genes, while the development of carpels is governed by D-class (SEEDSTICK (STK) and SHATTERPROOF (SHP)) and E-class genes [17,18,19]. Many MADS-box genes have been reported to affect anther development and thus impact plant fertility. For example, loss of function of C-class MADS-box gene OsMADS3 leads to brown anthers and a male-sterile phenotype in rice [20]. Overexpression of B-class MADS-box gene BcAP3 causes abnormal development of the anther wall in A. thaliana, resulting in reduced pollen viability and ultimately leading to male sterility [21]. A recent study showed that overexpression of B-class MADS-box gene PbTM6a in tomato reduced pollen viability, and germination thus caused male sterility [22]. These highlight the importance of MADS-box genes in the regulation of male reproductive development in plants.
Salvia miltiorrhiza Bunge, commonly known as Danshen, is a perennial plant species native to China and has been used in traditional Chinese medicine for over 2000 years [23]. It is known for its medicinal properties and has been used to treat a variety of conditions, including cardiovascular disease, cancer, and various types of inflammation [24]. Moreover, due to its short life cycle, strong vitality, mature transgenic technology, small genome, and low chromosome numbers, S. miltiorrhiza is considered an ideal model medicinal plant [25,26,27]. Although S. miltiorrhiza is grown in many parts of China, there is a large variation in the yield of active ingredients among different areas, with the best production coming from Sichuan and Shandong province [28,29,30]. Notably, Sichuan ecotype (S. miltiorrhiza_SC) is rich in salvianolic acid B, while Shandong ecotype (S. miltiorrhiza_SD) is rich in tanshinone IIA [31,32]. Furthermore, the Sichuan ecotype exhibits male sterility [32,33]. However, it is still unclear whether MADS-box genes are involved in regulating the male sterility of S. miltiorrhiza.
In the present study, we identified MADS-box gene family members based on the S. miltiorrhiza (cv. Sichuan) genome data, and studied their phylogeny, gene structures, conserved motifs, gene duplication, collinearity, cis-acting elements, and interacting proteins. In addition, we investigated the gene expression profile of SmMADS genes at different anther developmental stages between two ecotypes of S. miltiorrhiza and identified differentially expressed genes (DEGs). The findings of our study will provide a comprehensive analysis of the MADS-box family members in S. miltiorrhiza_SC and shed light on their potential roles in regulating anther development and male sterility.

2. Results

2.1. Identification and Physicochemical Property Analysis of MADS-box Gene Famliy in S. miltiorrhiza_SC

Based on the hidden Markov model (HMM) of the SRF-TF domain (PF00319) and the K-box domain (PF01486), a total of 63 MADS-box family genes were found in S. miltiorrhiza_SC and named as SmMADS1-63 based on their chromosomal and physical locations (Table 1). Protein physical and chemical properties, including the length of protein sequence, the molecular weight (MW), the isoelectric point (pI), and the subcellular localization, were analyzed (Table 1). Among the 63 SmMADS proteins, SmMADS48 was identified as the shortest protein with 66 amino acid (aa), whereas the longest one was SmMADS4 with 427 aa. The MW of the proteins ranged from 7.63 to 48.57 kDa, and the pI ranged between 4.90 (SmMADS4) and 10.15 (SmMADS23). Predictions of subcellular localization showed that all SmMADS proteins were located in the nucleus.

2.2. Phylogenetic Analysis of the MADS-box Gene Family

A phylogenetic tree was constructed to clarify the evolutionary relationship of MADS-box proteins among 63 SmMADS and 108 AtMADS proteins. Based on the grouping of MADS-box family proteins in A. thaliana [10], the MADS-box proteins of S. miltiorrhiza_SC were classified into two types and 17 subfamilies (Figure 1). The type I (14) included three subfamilies: Mα (8), Mβ (3), and Mγ (3). The type II (49) included 14 subfamilies: MIKC* (7), AG/STK (6), AGL2 (1), AGL6 (4), AGL12 (1), AGL15 (2), AGL17 (5), AP1 (SQUA) (2), AP3 (DEF) (3), FLOWERING LOCUS C (FLC) (TM3) (4), GGM13 (Bsister) (1), PI (GLO) (1), SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1) (6), and SHORT VEGETATIVE PHASE (SVP) (STMADS11) (6). Numbers in brackets represent the number of genes contained in each subfamily. Notably, AGL6, SVP, and AP3 subfamilies are significantly expanded in S. miltiorrhiza_SC compared with A. thaliana, and Mα, Mβ, Mγ, AGL2, and AP1 subfamilies are significantly contracted.

2.3. Chromosomal Distribution, Gene Structure, and Conserved Motif Analysis of SmMADS

In total, 63 SmMADS genes were unevenly spread over the eight chromosomes (Figure 2). Chromosome 6 had the maximum number of SmMADS genes (14), while chromosome 2 had the lowest number (2) (Table 1 and Figure 2). It can be seen that the majority of SmMADS genes were located at both ends of the chromosomes.
Gene structure analysis showed that the number of exons of SmMADS genes ranged from 1 to 12 (Table 1 and Figure 3A). Among type I SmMADS genes, 12 genes (SmMADS1/2/9/19/22/27/50/51/52/54/59/62) have only one exon and the other two genes SmMADS13 and SmMADS23 have two and three exons, respectively. Among type II SmMADS genes, except for SmMADS48 which has only two exons, the remaining 48 genes have 5 to 12 exons. It is obvious that the average number of exons in type II (7.6) is significantly higher than that in type I (1.2). Conserved motif analysis found a total of 10 conserved motifs, which are named motifs 1 to 10 (Figure 3B and Table S1). All SmMADS proteins had the conserved MADS-box domain (Motif 1 and 3) at the N-terminal, and K-domain (Motif 2, 4, and 7) was only found in type II SmMADS proteins.

2.4. Duplication and Synteny Analysis of SmMADS

Gene duplication events mainly includes three categories: whole genome duplication (WGD), segmental duplication, and tandem duplication [34]. Among SmMADS genes, a total of six genes (9.52%) were found to form three tandem duplication gene pairs, and 23 genes (36.51%) formed 14 segmental duplication gene pairs (Figure 4 and Table S2). It can be seen that segmental duplication is the main force for the expansion of the SmMADS gene family. Then, we calculated Ka/Ks ratios to investigate the evolutionary pressures on the orthologous MADS-box gene pairs, we found that all gene pairs exhibited Ka/Ks <1 (Table S2), indicating that SmMADS genes were purified by selection and to mitigate harmful mutations. To further analyze the orthologous relationships between SmMADS genes and those of other species, seven species were subjected to synteny analysis, including monocotyledon plants: O. sativa, and Z. mays, dicotyledon plants: A. thaliana, Sesamum indicum, Scutellaria baicalensis, S. bowleyana, and S. splendens. A total of 445 pairs of orthologous genes were identified (Table S3). Among them, the highest number of collinear gene pairs (178) were found between S. miltiorrhiza_SC and S. splendens, followed by S. baicalensis (79), S. bowleyana (72), A. thaliana (48), S. indicum (45), O. sativa (14), and Z. mays (9). It can be assumed that the collinearity of SmMADS genes is more significant between dicotyledons. Furthermore, SmMADS4, SmMADS5, and SmMADS16 have collinear relationships with all seven species, which indicates that they have retained ancestral functions.

2.5. Cis-Acting Elements Analysis of SmMADS

To gain insights into the regulatory mechanisms of SmMADS gene expression, cis-acting elements were analyzed (Figure 5). The results revealed that many cis-acting elements related to plant growth and development, plant hormones, and stress response, we classified them into two types. Type I cis-acting elements are mainly related to stress response (Figure 5A), including light responsive elements (AT1-motif, ATC-motif, ATCT-motif, etc.), anaerobic responsive element (ARE), drought responsive element (DRE), low temperature responsive element (LTR), and wound responsive element (WRE3 and WUN-motif). Type II cis-acting elements are mainly related to plant hormones and transcription factor binding sites (Figure 5B), including salicylic acid responsive elements (as-1, TCA-element, and SARE), abscisic acid responsive elements (ABRE), auxin responsive elements (AuxRR and TGA), MeJA-responsive elements (CGTCA-motif and TGACG-motif), ethylene responsive elements (ERE), gibberellin responsive elements (F-box, GARE-motif, P-box, and TATC-box), MYB binding sites (MYB, CCAAT-box, MBS, and MBSI), MYC binding site (MYC), and HD-ZIP binding site (HD-Zip).

2.6. Protein–Protein Interaction Network of SmMADS

To understand the interaction relationships and biological functions among SmMADS proteins, protein–protein interaction network was predicted based on MADS-box homologous gene in A. thaliana. A total of 36 SmMADS proteins homologous to those in A. thaliana and 15 corresponding interacting functional genes were identified (Figure 6). Among them, AG (SmMADS21/26/38/39), AP3 (SmMADS31/32/33), PI (SmMADS63), AGL2 (SmMADS12), AGL8 (SmMADS6/45), AGL20 (SmMADS20/46/61), AGL24 (SmMADS24), TT16 (SmMADS40), and STK (SmMADS41/56) might participate in flower development. SVP (SmMADS5/15/16/25/57), FLC (SmMADS29), MAF1 (SmMADS34/35/36), and AGL15 (SmMADS60) might participate in regulating flowering. AGL61 (SmMADS19/22/51/54) and AGL80 (SmMADS1/2/13/27) might participate in ovule development.

2.7. SmMADS Gene Expression during Anther Development

S. miltiorrhiza_SC displays male sterility, with its stamens being shorter compared to those of S. miltiorrhiza_SD (Figure 7A). At the mature pollen stage, the anthers of S. miltiorrhiza_SD normally opened and released a large number of mature pollen grains, while those of S. miltiorrhiza_SC did not split or only released a very small amount of non-viable pollen grains (Figure 7B). Additionally, the pollen grains of S. miltiorrhiza_SD were fusiform or subspherical, whereas those of S. miltiorrhiza_SC tended to stick together and exhibited irregular shapes (Figure 7C).
In order to investigate the potential role of SmMADS genes in anther development and male sterility, we selected the anther of S. miltiorrhiza_SC and S. miltiorrhiza_SD (fertile) at three different developmental stages for RNA-seq. The transcriptome data of SmMADS genes showed high correlation between each replicate (Figure S1). Based on the FPKM data, we found that the expression level of type II SmMADS genes are significantly higher than type I (Table S4 and Figure 8A). It is worth noting that genes of AP3 (SmMADS31/32/33), PI (SmMADS63), and AG (SmMADS21/26/38/39) subfamilies were all highly expressed during anther development in two ecotypes of S. miltiorrhiza. Interestingly, these genes all belonged to the B-class (AP3/PI) and C-class (AG) MADS-box genes according to the ABCDE model [18].
We also analyzed DEGs in the two ecotypes of S. miltiorrhiza at three different anther developmental stages (Figure 8B). Significant differential expression (|log2 Fold change| > 1) of 25 SmMADS genes (3 genes at MEI stage, 11 genes at YM stage, and 11 genes at MP stage) was observed in S. miltiorrhiza_SC as compared to S. miltiorrhiza_SD. Among them, SmMADS11/12 were upregulated and SmMADS27 was downregulated at MEI stage, SmMADS11/13/29/54/62 were upregulated and SmMADS5/7/10/27/35/47 were downregulated at YM stage, and SmMADS5/11/12/13/29/35/36/39 were upregulated and SmMADS6/18/44 were downregulated at MP stage.

3. Discussion

3.1. Identification and Classification of MADS-box Genes in S. miltiorrhiza_SC

With the completion of whole genome sequencing, the MADS-box gene family has been extensively studied in various plants, such as A. thaliana (108) [10], rice (75) [11], maize (75) [12], grape (83) [13], alfalfa (120) [14], cucumber (43) [35], lettuce (82) [36], and pear (95) [37]. In this study, 63 SmMADS genes were identified in the male sterility ecotype S. miltiorrhiza_SC, which is different from the 72 genes found in the male fertility ecotype S. miltiorrhiza_SD [38]. They were both divided into two types and 17 subfamilies according to phylogenetic relationship. Similar to many plants, such as rice, grape, lettuce, and pear [11,13,36,37], the number of type II genes was higher than type I. The gene structure analysis showed that the number of introns in type II genes was significantly higher than type I. As introns are involved in various steps of mRNA processing, including transcription, translation, and mRNA decay, they can have a significant impact on gene expression [39,40]. Thus, the regulating mechanism of type II genes may be more complex than type I. This phenomenon was also found in A. thaliana, rice, and pear [11,12,37]. Moreover, protein motif analysis showed that all SmMADS proteins had the highly conserved MADS domain, whereas type II genes contained unique I, K, and C domains, facilitating the formation of dimers and higher-order complexes between MADS-box proteins and transcriptional activation [6,7,8,9]. Therefore, type II SmMADS proteins have more complex protein structures, which also suggests that their regulatory mechanism may be more complex than type I. Additionally, we identified cis-acting elements in the promoter region of SmMADS genes that are associated with plant growth and development, plant hormones, and stress response, which was not analyzed in S. miltiorrhiza_SD [38]. These cis-acting elements play a crucial role in regulating the expression of related genes [40], enhancing the ability of S. miltiorrhiza to adapt to various adverse environments and ensure its normal growth and development.

3.2. Evolutionary Analysis of SmMADS Gene Family

Gene duplication events play an important role in organismal evolution, which provides a genetic basis for the emergence of new traits [41,42,43]. These events mainly include WGD, segmental duplication, and tandem duplication [34]. In this study, 17 duplication gene pairs were found in SmMADS genes, and the majority of these pairs (82.35%) were found to be segmental duplications, which was lower than S. miltiorrhiza_SD (19 and 84.21%) [38]. This indicates that segmental duplication is the main force for the expansion of the SmMADS gene family, which is consistent with findings in other species, such as Fagopyrum tataricum [44], Rhododendron hainanense Merr. [45], and Sechium edule [46]. The WGD, as the driving force of the expansion of gene families [47], was not identified in the SmMADS gene family, which might be the reason for the relatively small number of SmMADS genes. Moreover, we calculated the Ka/Ks ratio between these duplicated gene pairs and found that all ratios were all less than 1, which suggested that these duplication gene pairs were under purifying selection, which reduces genetic diversity [41]. This observation also implies that they are relatively conserved and less diverged. Purifying selection typically contributed to the functional redundancy. Our results demonstrate that these duplication gene pairs have similar conserved motifs and gene expression patterns, which indicates that their function may therefore be redundant. These findings suggest that the MADS-box family is highly conserved in the evolution of S. miltiorrhiza. Synteny analysis is a powerful tool for understanding the evolutionary trajectory of genes [48]. In this study, some collinear gene pairs were only identified between S. miltiorrhiza_SC and dicotyledon plants, indicating that these homologous pairs were formed after the differentiation of dicotyledonous and monocotyledonous plants. Additionally, some collinear gene pairs were identified between S. miltiorrhiza_SC and all seven other species, indicating that these homologous pairs may have existed before the divergence of their common ancestor. These phenomena were also observed in S. miltiorrhiza_SD, although the analysis was limited to the collinearity between S. miltiorrhiza_SD, A. thaliana, and rice [38]. In summary, these results indicate that the MADS-box gene family in S. miltiorrhiza exhibits evolutionary conservation.

3.3. MADS-box Genes May Participate in Regulating the Anther Development and Male Fertility of S. miltiorrhiza

The anther, together with the filament, constitutes the male reproductive organs of flowering plants and is where pollen development occurs [49]. The morphology and development of the anther are closely associated with the fertility of plants [50]. Previous studies have shown that MADS-box genes are crucial in anther development and male fertility. For example, the mutation of B-class and C-class MADS-box genes, such as OsMADS3 [20], BcAP3 [21], PbTM6a [22], FaTM6 [51], and OsMADS58 [52], can cause abnormal anther development and lead to male infertility. Gene expression profiling is important for determining gene function and biological processes. Most of the B-class and C-class MADS-box genes exhibited high expression levels in the stamen of S. miltiorrhiza_SD, indicating their significant roles in stamen development [38]. Our study demonstrated that all B-class (SmMADS31/32/33/63) and C-class (SmMADS21/26/38/39) genes were highly expressed during anther development in two ecotypes of S. miltiorrhiza, suggesting their crucial roles in anther development. Protein–protein interaction network revealed that there is an interaction between the B-class and C-class SmMADS proteins, suggesting that they do not act independently but form a complex to co-regulate the development of anthers.
Comparing DEGs in the transcriptomes of normal and male-sterile anthers is of great significance for identifying key genes underlying male sterility. In this study, SmMADS11, which belongs to the AGL6-like subfamily, was differentially expressed at all three anther development stages. Although not included in the traditional ABCDE model, AGL6-like genes play an important role in floral organ development [53]. For example, AGL6-like gene AGL13 is involved in regulating the formation of male and female gametophytes in A. thaliana [54]. In maize and rice, AGL6-like genes ZAG3 and OsMADS6 both regulate the development of stamens and ovules [55,56]. In wheat, RNAi of AGL6 results in the abnormal development of stamens and ovules [57]. Therefore, differential expression of SmMADS11 in anthers may affect normal anther development and result in male sterility. We will investigate the functional aspects of this gene in a further study.
Overall, the above findings provide insight into the potential functional roles of SmMADS genes in regulating the anther development and male fertility of S. miltiorrhiza.

4. Materials and Methods

4.1. Identification of MADS-box Genes in S. miltiorrhiza_SC

The genomic data of Sichuan S. miltiorrhiza (S. miltiorrhiza_SC) were published by our team and were publicly accessible at NCBI (https://www.ncbi.nlm.nih.gov/, accessed on 27 July 2022). The HMM profiles of the SRF-TF domain and the K-box domain were downloaded from the Pfam database (http://pfam.xfam.org/search, accessed on 11 January 2022). The HMMER version 3.3.1 software (http://hmmer.org/download.html, accessed on 12 January 2022) was utilized to identify the MADS-box gene family (E-value ≤ 10–10) [58]. The SMART program (http://smart.embl-heidelberg.de/, accessed on 20 January 2022) and Plant TFDB (http://planttfdb.gao-lab.org/index.php, accessed on 22 January 2022) were used to further ensure the accuracy of the screening results. In addition, physicochemical properties of SmMADS proteins, including the physical location, the molecular weights (MW), and theoretical isoelectric points (pI) were evaluated using the online ProtParam tool (http://web.expasy.org/protparam/, accessed on 11 August 2022). Subcellular localization prediction was carried out by using the Plant-mPloc server (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi, accessed on 11 August 2022).

4.2. Phylogenetic Analysis of SmMADS Genes

The MADS-box protein sequence of A. thaliana was downloaded from The Arabidopsis Information Resource (TAIR) database (http://www.arabidopsis.org/, accessed on 6 May 2021). Full-length amino acid sequences of the MADS-box protein of S. miltiorrhiza_SC and A. thaliana were aligned by Clustal W with default parameters [59]. A phylogenetic tree estimation under the maximum likelihood (ML) principle was constructed by MEGA X version 10.2.6 software with the neighbor-joining algorithm and default settings [60]. The evolutionary distances were calculated using the Poisson model. The phylogeny was tested by bootstrapping with 1000 replications. Finally, the ML tree was visualized by iTOL version 6 (https://itol.embl.de/, accessed on 12 March 2022) [61].

4.3. Chromosomal Localization, Gene Structure, and Conserved Motif Analysis

The distribution of SmMADS genes and gene density were extracted and visualized from the genome structure annotation (Gff file) using TBtools version 0.396 [62]. The exon–intron structure of SmMADS genes was constructed by the Gene Structure Display Server (GSDS) version 2.0 (http://gsds.cbi.pku.edu.cn/index.php, accessed on 21 March 2022) based on the Gff file [63]. MEME Suite version 5.5.0 (http://memesuite.org/tools/meme, accessed on 20 March 2022) [64] was employed to analyze the conserved motifs of SmMADS protein sequences. The maximum number of motifs was 10, the motif width ranged from 6 to 200 amino acid residues, and the screening threshold was E < e-10. Gene structure and conserved motifs were visualized by TBtools.

4.4. Duplication and Synteny Analysis

Gene duplication analysis was performed by using Clustal W to compare coding sequences (CDS) of SmMADS genes. Gene duplication events were defined and included the following: the aligned region with a similarity above 75%, the length difference of sequences no more than 25%, and only 1 duplication event is counted for tightly linked genes. Multiple collinear scanning toolkit (MCScanX) [65] was utilized to analyze the collinear blocks of MADS-box genes across different species and visualized by TBtools. Synonymous (Ka) and nonsynonymous (Ks) substitutions, as well as their ratios, were calculated by KaKs_Calculator [66].

4.5. Cis-Element and Protein–Protein Interaction Network Analysis

The putative promoter sequence regions 2000 bp upstream of each SmMADS gene was extracted as the promoter sequence, cis-acting elements were predicted using PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 2 April 2022) and visualized by TBtools. The SmMADS protein interaction network was examined using the online website String version 11.5 (https://string-db.org/, accessed on 12 August 2021) and visualized by Cytoscape version 3.9.1 software [67].

4.6. Plant Materials, Expression Profile Analysis of SmMADS Genes

Stamens and anthers of S. miltiorrhiza_SC and S. miltiorrhiza_SD were collected from Zhongjiang, Sichuan Province. Paraffin sections were created according to Wang et al. (2015) [68]. In brief, the specimens were dehydrated using a graded ethanol series and embedded in paraffin (Hualing, Shanghai, China). The 5 µm thick cross-sections were placed on gelatin-coated slides (Solarbio, Beijing, China) and stained with safranine and fast green double dyeing (Solarbio, Beijing, China). Then, paraffin sections of anthers were observed using Olympus BX51 microscope (Olympus, Tokyo, Japan).
The RNA-seq data of anther of S. miltiorrhiza_SC and S. miltiorrhiza_SD at different anther developmental stages published by our team [33] (publicly accessible at NCBI, https://www.ncbi.nlm.nih.gov/, accessed on 28 July 2022) were used to explore the expression patterns and differentially expressed genes of SmMADS genes during meiosis stage (MEI), young microspore stage (YM), and mature pollen stage (MP). Gene expression levels were estimated using fragments per kilobase of transcript per million fragments sequenced (FRKM). Differential gene expression analysis between S. miltiorrhiza_SC and S. miltiorrhiza_SD was performed using the DEGSeq R package. A corrected p-value of 0.05 and |log2 (Fold change)| of 1 were set as the criteria for significant differential gene expression. The expression matrix was visualized using TBtools.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/ijms241310937/s1.

Author Contributions

S.C. and K.L. performed the experiments, collected the data, and wrote the manuscript. X.D., L.W., and Y.J. participated in experiment implementation and data analysis. J.L. and R.Y. visualized graphics and revised manuscripts. L.Z. led the research, provided funding. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Modern Agricultural Industry Technology System Sichuan Innovation Team (SCCXTD-2020-19), Sichuan Science and Technology Program (2022ZHCG0095, 2023NSFSC1271, 2023NSFSC0663), Sichuan Crops and Animals Breeding Special Project (2021YFYZ0012) and Featured Medicinal Plants Sharing and Service Platform of Sichuan Province.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

S. miltiorrhiza (cv. Sichuan) genome is available from the NCBI under project ID PRJNA862689. RNA-seq data are available from the NCBI under project ID PRJNA863332.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Phylogenetic tree of S. miltiorrhiza_SC-A. thaliana MADS-box transcription factors. Different subfamilies of proteins are highlighted with different colors. The red and gray circles represent S. miltiorrhiza_SC and A. thaliana, respectively.
Figure 1. Phylogenetic tree of S. miltiorrhiza_SC-A. thaliana MADS-box transcription factors. Different subfamilies of proteins are highlighted with different colors. The red and gray circles represent S. miltiorrhiza_SC and A. thaliana, respectively.
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Figure 2. Chromosomal distribution of the MADS-box family members of S. miltiorrhiza_SC.
Figure 2. Chromosomal distribution of the MADS-box family members of S. miltiorrhiza_SC.
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Figure 3. Gene structure and protein motif of SmMADS genes. (A) Phylogenetic tree of SmMADS proteins. (B) Gene structure of SmMADS genes. (C) Conserved motif of SmMADS proteins.
Figure 3. Gene structure and protein motif of SmMADS genes. (A) Phylogenetic tree of SmMADS proteins. (B) Gene structure of SmMADS genes. (C) Conserved motif of SmMADS proteins.
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Figure 4. Synteny analysis of MADS-box genes between S. miltiorrhiza_SC and other plant species. (A) Synteny analysis of MADS-box genes in S. miltiorrhiza_SC. (B) Synteny analysis of MADS-box genes between S. miltiorrhiza_SC and A. thaliana, O. sativa, Z. mays, S. bowleyana, S. indicum, S. baicalensis, and S. splendens. The gray lines in the background indicate the collinear blocks within the genome, while the red lines highlight the syntenic of MADS-box gene pairs.
Figure 4. Synteny analysis of MADS-box genes between S. miltiorrhiza_SC and other plant species. (A) Synteny analysis of MADS-box genes in S. miltiorrhiza_SC. (B) Synteny analysis of MADS-box genes between S. miltiorrhiza_SC and A. thaliana, O. sativa, Z. mays, S. bowleyana, S. indicum, S. baicalensis, and S. splendens. The gray lines in the background indicate the collinear blocks within the genome, while the red lines highlight the syntenic of MADS-box gene pairs.
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Figure 5. Predicted cis-elements in the promoter regions of the SmMADS genes. (A) Cis-elements related stress response. (B) Cis-elements related plant hormones and transcription factor binding sites.
Figure 5. Predicted cis-elements in the promoter regions of the SmMADS genes. (A) Cis-elements related stress response. (B) Cis-elements related plant hormones and transcription factor binding sites.
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Figure 6. Protein–protein interaction network of SmMADS proteins based on their orthologs in A. thaliana.
Figure 6. Protein–protein interaction network of SmMADS proteins based on their orthologs in A. thaliana.
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Figure 7. Morphologies of stamens and anthers of S. miltiorrhiza_SC and S. miltiorrhiza_SD at mature pollen stage. (A) Morphologies of stamens. (B) Morphologies of anthers. (C) Transverse sections of anthers; 1–2: S. miltiorrhiza_SC, 3–4: S. miltiorrhiza_SD, EP: epidermis, EN: drug chamber inner wall, PG: pollen grains.
Figure 7. Morphologies of stamens and anthers of S. miltiorrhiza_SC and S. miltiorrhiza_SD at mature pollen stage. (A) Morphologies of stamens. (B) Morphologies of anthers. (C) Transverse sections of anthers; 1–2: S. miltiorrhiza_SC, 3–4: S. miltiorrhiza_SD, EP: epidermis, EN: drug chamber inner wall, PG: pollen grains.
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Figure 8. Expression profiles of SmMADS genes. (A) Expression profile of SmMADS genes in S. miltiorrhiza_SC and S. miltiorrhiza_SD at different stages of anther development. Gene expression level is shown on a graded color scale based on log2(FPKM + 1) values. (B) The ratio of the expression levels of SmMADS genes between S. miltiorrhiza_SC and S. miltiorrhiza_SD at different stages of anther development. The ratio of the expression levels is presented as log2 fold change (log2 FC) of the mean FPKM. SC: Sichuan; SD: Shandong; MEI: meiosis stage; YM: young microspore stage; MP: mature pollen stage.
Figure 8. Expression profiles of SmMADS genes. (A) Expression profile of SmMADS genes in S. miltiorrhiza_SC and S. miltiorrhiza_SD at different stages of anther development. Gene expression level is shown on a graded color scale based on log2(FPKM + 1) values. (B) The ratio of the expression levels of SmMADS genes between S. miltiorrhiza_SC and S. miltiorrhiza_SD at different stages of anther development. The ratio of the expression levels is presented as log2 fold change (log2 FC) of the mean FPKM. SC: Sichuan; SD: Shandong; MEI: meiosis stage; YM: young microspore stage; MP: mature pollen stage.
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Table
Table 1. Physicochemical properties of SmMADS proteins.
Table 1. Physicochemical properties of SmMADS proteins.
Gene NameGene IDGene StartGene EndProtein LengthMW (kDa)pIExonSubcellular Localization
SmMADS1SmiChr010159.11,485,3621,486,00921524.159.441Nucleus
SmMADS2SmiChr010237.12,485,7292,486,46324426.918.781Nucleus
SmMADS3SmiChr010463.15,985,4255,987,88026729.086.028Nucleus
SmMADS4SmiChr012432.147,986,87147,991,14342748.574.9011Nucleus
SmMADS5SmiChr012518.149,853,73749,858,25122825.795.747Nucleus
SmMADS6SmiChr013041.157,213,03757,237,11836741.278.7611Nucleus
SmMADS7SmiChr013114.157,973,95957,977,15542148.545.4012Nucleus
SmMADS8SmiChr013140.158,250,72458,253,61642148.195.2410Nucleus
SmMADS9SmiChr013188.158,984,44458,984,92916118.939.071Nucleus
SmMADS10SmiChr013568.163,166,26263,170,46024928.748.829Nucleus
SmMADS11SmiChr020206.11,687,8381,692,12921424.829.598Nucleus
SmMADS12SmiChr022579.150,749,43350,753,42125529.216.718Nucleus
SmMADS13SmiChr030457.16,931,5866,932,57623026.159.082Nucleus
SmMADS14SmiChr032079.139,590,71139,600,91625329.359.238Nucleus
SmMADS15SmiChr032342.143,073,81943,078,12721424.086.867Nucleus
SmMADS16SmiChr032371.143,524,70043,529,89223025.946.948Nucleus
SmMADS17SmiChr032483.145,482,82045,488,96125028.438.906Nucleus
SmMADS18SmiChr032484.145,496,31445,498,20032336.595.3811Nucleus
SmMADS19SmiChr033147.153,409,97153,410,47416719.268.361Nucleus
SmMADS20SmiChr040450.110,096,93310,103,41520823.979.107Nucleus
SmMADS21SmiChr042485.149,933,46949,936,88624427.879.077Nucleus
SmMADS22SmiChr042537.150,422,60050,423,25321724.316.401Nucleus
SmMADS23SmiChr042539.150,444,33250,450,33121724.1110.153Nucleus
SmMADS24SmiChr050234.14,973,8894,978,37920122.735.497Nucleus
SmMADS25SmiChr050236.14,985,2894,990,24015016.839.485Nucleus
SmMADS26SmiChr050368.16,663,0016,667,86024628.409.577Nucleus
SmMADS27SmiChr051941.124,790,93124,791,80329033.849.311Nucleus
SmMADS28SmiChr052086.126,494,69426,506,51422826.328.268Nucleus
SmMADS29SmiChr052419.130,819,76730,835,51419722.365.817Nucleus
SmMADS30SmiChr052857.136,559,13336,566,90820724.329.417Nucleus
SmMADS31SmiChr053058.139,835,85339,837,66020523.939.205Nucleus
SmMADS32SmiChr053148.141,413,13841,415,04024128.189.607Nucleus
SmMADS33SmiChr053244.143,027,13843,029,14921024.565.116Nucleus
SmMADS34SmiChr054722.175,497,64775,508,99319121.786.767Nucleus
SmMADS35SmiChr055132.180,642,42480,653,56118721.138.217Nucleus
SmMADS36SmiChr055133.180,660,60780,663,67219522.458.207Nucleus
SmMADS37SmiChr060209.14,683,1054,688,85420623.408.976Nucleus
SmMADS38SmiChr061372.135,548,75835,554,00125129.439.087Nucleus
SmMADS39SmiChr061672.141,801,64541,807,13924428.179.027Nucleus
SmMADS40SmiChr061960.146,937,82146,939,44224028.006.846Nucleus
SmMADS41SmiChr062294.152,175,55552,180,24022425.949.478Nucleus
SmMADS42SmiChr062306.152,366,57452,369,92521524.688.287Nucleus
SmMADS43SmiChr062755.158,203,25858,205,95433538.566.029Nucleus
SmMADS44SmiChr063526.168,122,35968,129,35923226.779.217Nucleus
SmMADS45SmiChr063695.169,688,07369,700,71227030.906.649Nucleus
SmMADS47SmiChr063823.171,111,09171,114,80725628.857.758Nucleus
SmMADS48SmiChr063826.171,136,93371,137,349667.6310.012Nucleus
SmMADS49SmiChr063828.171,151,76471,155,53825628.857.758Nucleus
SmMADS50SmiChr063850.171,360,77371,361,51924828.949.441Nucleus
SmMADS51SmiChr070750.18,813,1208,813,79122324.899.341Nucleus
SmMADS52SmiChr070751.18,816,9618,817,77627128.867.801Nucleus
SmMADS53SmiChr070801.19,225,5279,237,09123827.388.878Nucleus
SmMADS54SmiChr070926.110,535,23110,536,10329031.828.201Nucleus
SmMADS55SmiChr073037.137,186,39937,197,55121124.109.227Nucleus
SmMADS56SmiChr073378.142,810,56642,813,27821925.219.578Nucleus
SmMADS57SmiChr074083.153,784,25953,791,38920022.819.669Nucleus
SmMADS58SmiChr074093.153,967,20353,977,68423927.589.389Nucleus
SmMADS59SmiChr074108.154,295,91954,296,61423125.545.751Nucleus
SmMADS60SmiChr074429.160,910,55560,913,90126029.328.658Nucleus
SmMADS61SmiChr082309.155,443,03855,453,57321124.189.498Nucleus
SmMADS62SmiChr082932.162,821,91262,822,75127930.569.341Nucleus
SmMADS63SmiChr083047.163,750,69263,752,52521224.936.517Nucleus
MW: molecular weight, pI: isoelectric point.
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MDPI and ACS Style

Chai, S.; Li, K.; Deng, X.; Wang, L.; Jiang, Y.; Liao, J.; Yang, R.; Zhang, L. Genome-Wide Analysis of the MADS-box Gene Family and Expression Analysis during Anther Development in Salvia miltiorrhiza. Int. J. Mol. Sci. 2023, 24, 10937. https://doi.org/10.3390/ijms241310937

AMA Style

Chai S, Li K, Deng X, Wang L, Jiang Y, Liao J, Yang R, Zhang L. Genome-Wide Analysis of the MADS-box Gene Family and Expression Analysis during Anther Development in Salvia miltiorrhiza. International Journal of Molecular Sciences. 2023; 24(13):10937. https://doi.org/10.3390/ijms241310937

Chicago/Turabian Style

Chai, Songyue, Kexin Li, Xuexue Deng, Long Wang, Yuanyuan Jiang, Jinqiu Liao, Ruiwu Yang, and Li Zhang. 2023. "Genome-Wide Analysis of the MADS-box Gene Family and Expression Analysis during Anther Development in Salvia miltiorrhiza" International Journal of Molecular Sciences 24, no. 13: 10937. https://doi.org/10.3390/ijms241310937

APA Style

Chai, S., Li, K., Deng, X., Wang, L., Jiang, Y., Liao, J., Yang, R., & Zhang, L. (2023). Genome-Wide Analysis of the MADS-box Gene Family and Expression Analysis during Anther Development in Salvia miltiorrhiza. International Journal of Molecular Sciences, 24(13), 10937. https://doi.org/10.3390/ijms241310937

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