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Article

Genome-Wide Identification and Expression Analysis of the MADS-Box Gene Family in Cassava (Manihot esculenta)

by
Qin Zhang
1,†,
Yanan Li
1,†,
Sha Geng
1,
Qian Liu
1,
Yingchun Zhou
1,
Shaobin Shen
1,
Zhengsong Shen
1,
Dongxiao Ma
1,
Mingkun Xiao
1,
Xin Luo
1,
Bin Che
1,
Kang Li
1,2 and
Wei Yan
1,*
1
Institute of Tropical and Subtropical Cash Crops, Yunnan Academy of Agricultural Sciences, Baoshan 678000, China
2
Changning County Lan Hui Agricultural Development Company Limited, Baoshan 678000, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Horticulturae 2024, 10(10), 1073; https://doi.org/10.3390/horticulturae10101073
Submission received: 5 September 2024 / Revised: 30 September 2024 / Accepted: 7 October 2024 / Published: 8 October 2024
(This article belongs to the Section Genetics, Genomics, Breeding, and Biotechnology (G2B2))
Browse Figures
Versions Notes

Abstract

:
The MADS-box gene family constitutes a vital group of transcription factors that play significant roles in regulating plant growth, development, and signal transduction processes. However, research on the MADS-box genes in cassava (Manihot esculenta) has been relatively limited. To gain deeper insights into the functions of the MADS-box genes in cassava development, in this study, we undertook a comprehensive genome-wide identification of the MADS-box gene family in cassava. We identified a total of 86 MADS-box genes with complete domains in the cassava genome, designated as MeMADS01 to MeMADS86. Through bioinformatic analyses, we investigated the basic physicochemical properties, conserved motifs, chromosomal locations, and phylogenetic relationships of the cassava MADS-box genes. The MADS-box gene family of cassava exhibited conservation, as well as species-specific characteristics, with intron loss being a predominant mode of evolution for the MADS-box genes. Expression pattern variations in the MeMADS genes across different tissues offer insights into their potential functions. Time-ordered gene co-expression network (TO-GCN), transcriptome data, and RT-qPCR analysis suggested the responsiveness of the MADS-box genes to drought stress. Meanwhile, MeMADS12 might be involved in regulating flowering under drought conditions via an ABA (abscisic acid)-dependent pathway. These findings provide valuable resources for a deeper understanding of the biological roles of the MADS-box genes in cassava.

1. Introduction

The MADS-box gene family is widely found in plants and plays a key role in plant growth and development, including the regulation of the plant abiotic stress response, endosperm and embryonic development, seed germination, plant flowering transition, flowering time, flower development, and fruit formation [1,2,3,4]. According to the characteristics of protein domains and phylogenetic relationships, the MADS-box gene family can be classified into two types, namely, type I and type II [5], also known as SRF-like and MEF2 (myocyte enhancer factor 2)-like proteins, respectively [6]. The amino acid sequence of type I protein is encoded by approximately 180 nucleotides, including an M (Keratin-like)-type domain. Furthermore, it lacks a K-type domain [6,7]. Type II protein has not only an M-type domain but also other domains, such as a K-type domain, I (Intervening)-type domain, and C-terminal-type domain. According to phylogenetic relationships, type I can be classified into Mα, Mβ, and Mγ, and type II can be classified into MIKC* and MIKCC. Meanwhile, a phylogenetic analysis of MADS-box homologous genes in angiosperms showed that MIKCC includes 16 subfamilies, namely, AP1/FUL (APETALA1/FRUITFULL), AP3 (APETALA3), PI (PISTILLATA), AG (AGAMOUS), STK (SEEDSTICK), SEP (SEPALLATA), AGL6 (AGAMOUS-LIKE 6), AGL12, AGL15, AGL32, SVP (SHORT VEGETATIVE PHASE), OsMADS32, ANR1 (ARABIDOPSIS NITRATE REGULATED 1), SOC1/TM3 (SUPPRESSOR OF OVEREXPRESSION OF CO1), TM8 (TOMATO MADS-BOX 8), and FLC (FLOWERING LOCUS C) [6,8,9].
Manihot esculenta (M. esculenta) belongs to the family Euphorbiaceae, which is one of three major potato crops and the third largest food crop in hot areas. Moreover, M. esculenta has become the sixth most important economic food crop in the world because of its high starch content and excellent resistance to drought and poor soils. In addition, M. esculenta is an important source of raw materials for industrial starches, ethanol fuel, and green chemical materials, making it an economic crop with considerable development potential [10,11]. However, M. esculenta is mostly planted in tropical and subtropical arid areas, which are significantly affected by weather and environmental factors. As a result, it suffers from alternating droughts and floods, poor soil fertility, and other factors.
Drought is a major environmental factor that affects the geographical distribution and limits the reproduction of plants. An increasing amount of evidence from genetic and molecular analyses reveals that the MADS-box gene plays an important role in responding to drought stress. For example, the MADS-box gene SlMBP8 was silenced by RNA interference in tomato, resulting in the drought resistance of the mutant plant being significantly improved [12]. In the food crop rice, the transcription level of the OsMADS26 gene was downregulated using RNAi technology [13]. The drought resistance was improved and the expression of stress-related genes was upregulated in the rice after treatment. Meanwhile, the yield increased significantly compared with the wild type [14]. Research showed that OsMADS18, OsMADS22, OsMADS26, and OsMADS27 were upregulated more than twofold under drought stress, while OsMADS2, OsMADS30, and OsMADS55 were upregulated more than twofold under drought. Furthermore, OsMADS18 can interact with OsMADS6, OsMADS8, OsMADS7, and OsMADS47 under stress [14].
So far, the MADS-box gene family has not been studied systematically in M. esculenta, particularly the relationship between the MADS-box gene and drought stress. In this study, the MADS-box gene family in M. esculenta (MeMADS) was identified based on cassava genome and transcriptome data, and the basic characteristics of MeMADS gene family members were analyzed using bioinformatics methods. This will lay a foundation for further study on the function and mechanism of MeMADS in M. esculenta.

2. Materials and Methods

2.1. Materials

The cassava cultivars Arg7 and SC124 were obtained from the Institute of Tropical and Subtropical Cash Crops, Yunnan Provincial Academy of Agricultural Sciences (Baoshan 678000, China). For the drought treatment, 5-month-old potted cassava seedlings were placed in a photoperiod growth chamber (12 h light/12 h dark) and leaf and root tissue samples were collected 21 days after watering was ceased. A control group (CK) continued to receive normal watering. For the ABA treatment, 2 mL of 100 µmol·L−1 ABA was added to the culture bottles containing 3-month-old cassava plantlets. After placement in a photoperiod growth chamber, leaf samples were collected 12 h later. The samples were rapidly frozen in liquid nitrogen and then transferred to a −80 °C freezer for storage until use. Each sample was set to triplicate biological replicates.

2.2. Data Resources

The genome data of M. esculenta (Version = 8.1), Ricinus communis (R. communis), Hevea brasiliensis (H. brasiliensis), Jatropha curcas (J. curcas), Vernicia fordii (V. fordii), and Euphorbia lathyrism (E. lathyrism) were downloaded from the Euphorbiaceae Database (http://eupdb.liu-lab.com/, accessed on 10 May 2024). The genome data of Mercurialis annua (M. annua) and Vitis vinifera (V. vinifera) were downloaded from NCBI (https://www.ncbi.nlm.nih.gov/, accessed on 10 May 2024). The genome data of Populus trichocarpa (P. trichocarpa) and Salix purpurea (S. purpurea) were downloaded from the Phytozome database (https://phytozome-next.jgi.doe.gov/, accessed on 10 May 2024). Arabidopsis thaliana (A. thaliana) genome data were obtained from the Tair 10 database. The initial phylogenetic tree of 11 species was constructed using jolytree software (version = 2.1) and fixed through the Euphorbiaceae database [15].

2.3. Identification of the MADS-Box Gene Family

The profiles of the Hidden Markov Model (HMM) in the MADS-box gene family, PF00319 and PF01486, were obtained from the Pfam database. The putative MADS-box gene family members in each species were searched for using the hmmsearch program of HMMER 3.0 software, with the E-value set to 10−5. BLASTP (https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 12 May 2024) was used to screen the candidate’s MADS-box gene family. After removing the redundant genes, NCBI-CDD (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi, accessed on 12 May 2024) was used to filter candidate genes according to the conservative structure of the domain.

2.4. Phylogenetic Tree Construction in the MADS-Box Gene Family

Aligning MADS-box protein sequences through MAFFT software (version = 7.520), the parameter was set as follows: —maxiterate 1000—localpair [16]. Next, Trimal software (version 1.4) was used to trim the conserved region of the sequence using the following parameter: -automated1 —gappyout —gt 0.8 —st 0.001 —cons 60 [17]. IQ-TREE software (version = 2.2.6) was used to construct the phylogenetic tree via the maximum likelihood (ML) method, and the parameter was set to —m MFP —bb 1000 [18]. The MADS-box gene of A. thaliana was used to classify subfamilies in other species. The number of members of each subfamily in each species was counted.

2.5. Analysis of Basic Physicochemical Properties of the MADS-Box Gene Family in M. esculenta

ExPaSy online tools (https://web.expasy.org/compute_pi/, accessed on 12 May 2024) were used to estimate the molecular weight (MW) and theoretical isoelectric point (PI) [19]. Plant-PLoc (http://www.csbio.sjtu.edu.cn/bioinf/plant/, accessed on 12 May 2024) was used for subcellular localization prediction [20].

2.6. Motif and Gene Structure Analysis of the MADS-Box Gene Family in M. esculenta

In the prediction of the conservative motif of the MeMADS protein sequence through MEME software (https://meme-suite.org/meme/tools/meme, accessed on 14 May 2024), the maximum discovery number was set to 10 and the other parameters were kept as the default values [21].
FastTree (version = 2.1.11) software was used to construct the rootless phylogenetic tree of MeMADS members [22]. The conserved motifs and gene structures (exons/introns) were plotted using TBtools [23].

2.7. Chromosome Localization and Collinearity Analysis of the MADS-Box Gene Family in M. esculenta

The chromosome locations of MeMADS were obtained from the genome data of M. esculenta. Gene collinearity and homolinearity were analyzed using MCScanX [24]. The synonymous substitution rate (Ks) and non-synonymous substitution rate (Ka) were calculated through KaKs_Calculator [25].

2.8. Analyzing Cis-Acting Elements of the MADS-Box Gene Family Promoter in M. esculenta

TBtools software (version = v2.119) was used to extract the 2000 bp upstream sequence of MeMADS genes as the promoter. Then, the promoter sequence of MeMADS genes was submitted to the PlantCARE (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 16 May 2024) database to identify possible cis-acting element s [26].

2.9. Expression Analysis of the MADS-Box Gene Family in M. esculenta using RNA-Seq Analysis

All raw data from RNA-Seq were sourced from the NCBI database, including PRJNA324539 (transcriptome data for 11 tissues) [27], PRJNA491633 (transcriptome data for drought stress) [28], and PRJNA592177 (transcriptome data for cassava flowering during the dry season in the mountain region) [29].
For the transcriptome data of 11 tissues (PRJNA324539), cassava seedlings cultured in a greenhouse for 3 months were used to harvest various tissue samples, including leaf, petiole, stem, shoot apical meristem (SAM), fibrous roots, root apical meristem (RAM), and storage roots. In addition, the 4-week-old somatic organized embryogenic structure (OES) and the 3-week-old friable embryogenic callus (FEC) tissues of cassava plants were also sampled. Total RNA was isolated separately from the above samples for RNA-Seq analysis [27].
For the transcriptome data for drought stress (PRJNA491633), forty-five-day-old cassava seedlings were used as the research subjects, and drought stress was simulated using 20% polyethylene glycol (PEG). Full expanded leaves (FELs) and roots were collected at 0, 3, and 24 h after treatment for RNA-Seq analysis [28].
For the transcriptome data for cassava flowering (PRJNA592177), mature leaf samples were collected for RNA-Seq analysis on the following dates during the cultivation period: 31 May (05/31), 29 June (06/29), 26 July (07/26), 15 August (08/15), 11 September (09/11), 5 October (10/05), and 30 October (10/30) [29].
For the raw data above, fastQC (version = 0.12.0) software was first used for quality control and the low-quality reads were filtered using fastp [30]. Based on clean data, hisat2 [31] was used to map clean reads to the M. esculenta reference genome with default parameters. FeatureCounts (version = 2.0.3) [32] software was used to quantify gene expression, and the counts were measured by Fragments Per Kilobase of exon model per Million mapped fragments (FPKM). After normalization, the FPKM values were calculated as the logarithm with base 2 for plotting the heatmap.

2.10. Time-Ordered Gene Co-Expression Network (TO-GCN) Analysis

The construction of time-ordered gene co-expression networks followed the protocol available on the website (https://github.com/petitmingchang/TO-GCN_STAR-Protocol, accessed on 22 May 2024) [33]. The transcriptome data for cassava flowering (PRJNA592177) was used to construct the TO-GCN. Firstly, DESeq2 (version = 1.24.0) [34] was used to identify differentially expressed genes (DEGs) with the screening criteria |log2 (Fold change)| ≥ 1, p-value ≤ 0.05, and q-value < 0.05 [34,35,36]. After filtering, the DEGs were used to build the TO-GCN.

2.11. RNA Extraction and RT-qPCR Analysis

The cassava leaves were quickly ground in liquid nitrogen, and the total RNA was extracted using the RNAprep Pure Plant Total RNA Extraction Kit (TIANGEN BIOTECH Co., Ltd., Beijing, China). After extraction, the first-strand cDNA was synthesized according to the instructions provided with the Hifair® III Reverse Transcriptase Kit (Yeasen Biotechnology Co., Ltd., Shanghai, China). Primers were designed based on the transcript sequence of MeMADS12 (Manes.02G059300) (Supplementary Table S1), and the MeMADS12 was obtained via PCR and verified through sequencing for the subsequent analysis and experiments. Based on the MeMADS12 CDS sequence, specific quantitative primers were designed using NCBI-Primer-BLAST (Supplementary Table S1). The RT-qPCR analysis was performed in the LightCycler® 96 Real-Time PCR Detection System (Roche, Hercules, Switzerland) using Hieff UNICON® Universal Blue qPCR SYBR Green Master Mix (Yeasen Biotechnology Co., Ltd., Shanghai, China). The cassava β-actin gene (Manes.13G084300) was used as a reference gene (Supplementary Table S1). Data analysis was performed using the 2−ΔΔCT method, and significance testing for differences was conducted using IBM SPSS Statistics 25.0. Each sample was analyzed with three technical replicates. The primer pairs used for this study are provided in Supplementary Table S1.

2.12. Subcellular Localization of MeMADS12

SnapGene software (version = 6.0.2) was used to design the primer pairs according to the MeMADS12 CDS sequence and the restriction enzyme cutting site (Supplementary Table S1). Using the double-enzyme digestion method, the target gene was inserted into the plant overexpression vector pCAMBIA1300 to construct the pCAMBIA1300-MeMADS12-GFP vector. The primer pairs that were designed on the pCAMBIA1300 were used for the detection and verification of the recombinant vector (Supplementary Table S1). The recombinant plasmid and the pCAMBIA1300-GFP empty vector were transformed into competent LBA4404 Agrobacterium cells, which were injected into tobacco leaves. The localization of MeMADS12-GFP protein was observed under a laser confocal microscope (OLYMPUS FV1000) 3 days after the injection.

3. Results

3.1. Identification and Phylogenetic Analysis of the MADS-Box Gene Family in 11 Species

To explore the evolutionary relationship of the MADS-box gene in Malpighiales and Euphorbiaceae, the genome data of seven Euphorbiales, two Malpighiales, and two model plants were selected to identify the MADS-box gene family. Finally, an ML phylogenetic tree of 11 species was constructed (Figure 1). The subfamily classification and quantity statistics according to the MADS-box gene family members were identified in A. thaliana and V. vinifera. Ultimately, the number of MADS-box gene family members and the distribution of subfamily members in 11 species were plotted. Notably, previous study has shown that the OsMADS32 homologous gene is present in angiosperms and monocotyledonous plants, such as Amborella trichopoda, Oryza sativa, and Ananas comosus [8]. In this study, the homologous gene of OsMADS32 was not found in the genome data of 11 eudicot plants, which is consistent with the results of the previous study (Supplementary Table S2) [8].
In total, 86 MADS-box genes were identified in M. esculenta (Figure 1 and Figure 2, Supplementary Table S2). Among the seven Euphorbiaceae, M. annua has the largest number of MADS-box genes. However, its genome is not the largest among the seven species. In contrast, H. brasiliensis had the largest genome, while only 63 MADS-box genes were identified (Supplementary Table S2). Therefore, the number of MADS-box genes is probably independent of genome size for Euphorbiaceae. On the other hand, there are also differences in the number of genes and evolutionary patterns among different subfamilies. The MADS-box subfamilies of M. esculenta, V. fordii, R. communis, and M. annua are all preserved, which indicates evolutionary conservatism. However, J. curcas, H. brasiliensis, and E. lathyrism lack certain MADS-box gene subfamilies; for instance, J. curcas and H. brasiliensis each lack FLC, while E. lathyrism is missing six MADS-box subfamilies (Figure 1 and Figure 2, Supplementary Table S2).

3.2. Chromosome Localization and Physicochemical Property Analysis of Protein

Eighty-six MADS-box gene family members were identified in M. esculenta and were named according to the order of chromosomes (Figure 3). All 86 MeMADSs were distributed across 17 chromosomes, and no MADS-box genes were founded on chromosome 16 (Figure 3). Chromosome 02 and chromosome 05 had the largest number of MeMADSs with 10 each (Figure 3).
The results of the protein sequence analysis showed that the amino acid number, isoelectric point, and molecular weight of MeMADSs ranged from 87 to 379 aa, 4.69 to 10.92, and 9811.35 to 43,647.81 kDA, respectively (Supplementary Table S3). Seven MeMADSs were unstable proteins, namely, MeMADS9, MeMADS34, MeMADS29, MeMADS14, MeMADS63, MeMADS33, and MeMADS59. The other MeMADSs were stable proteins. All of the MeMADSs were hydrophilic proteins. The result of the subcellular localization prediction showed that all MeMADSs were located in the nucleus (Supplementary Table S3).

3.3. Analysis of Conserved Motif and Gene Structure of MeMADS-Box Protein

To further understand the structural diversity of the MeMADSs, analyses were conducted on their conserved motifs and gene structures (introns/exons) based on their phylogenetic relationships (Figure 4). An analysis of the conserved motifs revealed that the MeMADS proteins had 1 to 6 motifs (Figure 4b). All MeMADSs contained motif 1, a typical conserved structural domain in MeMADSs, namely, the M domain. Except for MeMADS17 and MeMADS83, all type II MeMADS proteins contained motif 5 and 8, which were conserved regions of the K-box domain (Figure 4b,c). The gene structure analysis showed that the longest sequence was found in MeMADS63 among all MeMADS genes. Most members of the MeMADS gene family of the same type or subfamily have similar gene structures. Nevertheless, a few members show variations in the arrangement of introns and exons, such as MeMADS65 and MeMADS16. All of these belonged to the type II subfamily (MIKC*). MeMADS16 had 10 exons, whereas MeMADS65 contained only 2 exons (Figure 4d). This could be due to changes in the number of introns during evolutionary processes. Furthermore, type I subfamilies generally had fewer exons and introns than type II, with most type I proteins containing only one or two exons and most type II proteins with at least seven exons (Figure 4d).

3.4. Analysis of Cis-Element of the MADS-Box Gene Promoter in M. esculenta

The promoter region upstream of a gene contains cis-elements that govern transcription. As depicted in Figure 5, these elements of MeMADS-box genes are classified based on their function into elements responsive to plant hormones, light, growth, stress, and essential promoter components (Figure 5, Supplementary Table S4). The light-responsive elements had the highest numbers in the MeMADS genes promoter, and all MeMADS gene promoter regions contained at least five light-responsive elements, with Box 4 being the most common. Furthermore, each MeMADS gene contained at least two cis-elements responsive to plant hormones. MeMADS genes exhibit an abundance of stress-responsive cis-elements, including LTR (a cis-acting element involved in low-temperature responsiveness) and MBS (an MYB binding site involved in drought inducibility) (Figure 5). In conclusion, MeMADS genes presumably play a crucial role in plant response to adversity and growth development.

3.5. Gene Duplication Analysis of the MADS-Box Gene Family in M. esculenta

Gene duplication is the primary driving force behind genome evolution [37]. Repeated genes usually evolve to distribute existing functions or acquire new functions to enhance plant adaptability [2]. In this study, we identified 15 pairs of paralogous genes, with all duplication types being WGD (whole-genome duplication) or segmental duplication. To analyze the selection pressure between MeMADS duplication genes, we analyzed the Ka, Ks, and Ka/Ks values for 15 pairs of paralogous genes. The results indicated that all gene pairs had a Ka/Ks ratio of less than 1, revealing that these gene pairs were under purifying selection and were likely functionally conserved (Table 1).

3.6. Analysis of MADS-Box Gene Expression Pattern in Tissues of M. esculenta via RNA-Seq Analysis

The FPKM values of MeMADSs were selected from the transcriptome data of 11 tissue samples for further analysis. Based on the results, it was found that different members of the MeMADS genes in cassava exhibit significant differences in expression levels across various tissues, which suggests that MeMADS genes may have diverse functions in the growth and development of cassava (Figure 6). MeMADS6 and MeMADS75 exhibited specific expression in the leaf and midvein, while MeMADS38/84/58/25/28 displayed specific expression in FEC. It was noteworthy that MeMADS60 and MeMADS67 have higher expression levels in the RAM, fibrous roots, and storage roots, suggesting that these transcription factors might play crucial roles in root development (Figure 6). Furthermore, MeMADS60 and MeMADS67 are part of the ANR1 group, a category of genes in A. thaliana that have been reported to be highly expressed in roots and to play a role in root development [38,39]. MeMADS67 is highly expressed in the storage root, implying that it likely functions as a negative regulator in the reactive oxygen species (ROS) signaling pathway. Its high expression is hypothesized to delay the deterioration of storage roots in cassava [40,41].

3.7. MADS-Box Gene Expression Pattern in M. Esculenta under Drought Stress via RNA-Seq Analysis

The FPKM values of the MeMADSs in leaves and roots were selected from the transcriptome data of drought-stressed cassava for further analysis. Here, 20% PEG was used to simulate drought treatment. After 3 h drought treatment, MeMADS12/13/22/24/72/80 were upregulated (Log2(Fold change) > 0.5) and MeMADS39/70 were downregulated (Log2(Fold change) < −0.5) in leaf (Figure 7). After 24 h of drought treatment, MeMADS22/29/72 were upregulated (Log2(Fold change) > 0.5) and MeMADS3/6/8/12/13/24/30/32/35/49/82 were downregulated (Log2(Fold change) < −0.5) in leaf (Figure 7). After 3 h of drought treatment, MeMADS15/30/32/59/67/72/80/81 were upregulated and MeMADS5/11/12 were downregulated (Log2(Fold change) < −0.5) in root (Figure 7). After 24 h of drought treatment, MeMADS35/63/80 were upregulated (Log2(Fold change) > 0.5) and MeMADS3/5/11/12/22/32/37/39/49/82/86 were downregulated (Log2(Fold change) < −0.5) in root (Figure 7).

3.8. Time-Ordered MADS-Box Gene Co-Expression in M. esculenta in Response to Flowering

Previous studies have shown that cassava flowering is induced in the dry season in mountain regions [29]. The MADS-box gene family plays a crucial role in regulating flower development and controls the timing of flowering [1,2,3]. To further understand the key genes in the cassava MADS-box gene family that affect flowering under drought-induced conditions during the dry season, we constructed a TO-GCN using the transcriptome data for cassava flowering in the mountain region during the dry season. In this analysis, the input consisted of the FPKM values of DEGs at seven time points (05/31, 06/29, 07/26, 08/15, 09/11, 10/05, and 10/30).
Using the TO-GCN program, a co-expression network was constructed between TF and non-TF genes (Figure 8). Finally, the result was generated with seven time-ordered levels. In the TO-GCN (Figure 8), the upper-level genes might be regulators of the genes co-expressed at the same or the next levels, and the latter might regulate certain co-expressed genes at the same or lower levels, etc., thus forming potential regulatory pathways with a clear hierarchical structure [42]. A total of 12 MeMADS transcription factors participated in the co-expression network. In the vegetative development phase (05/31–08/15, namely, Level 1–4), a total of six MeMADS genes participated in the regulatory network. At the full-bloom stage (Level 6–7), a total of six MeMADS transcription factors participated in the network regulation, and the MeMADS genes might participate in the development of flower organs and form mature organs at this stage (Figure 8). In addition, a TO-GCN of all TFs was constructed to investigate the co-expression relationship between MeMADSs and other TFs (Supplementary Figure S1). The results showed that A total of 11 MeMADS transcription factors participated in the co-expression network, while MeMADS82 did not show co-expression with other TFs.
To research the regulatory pattern of MeMADS gene expression for cassava flowering during the dry season in the mountain region, a subnetwork (Figure 9, Supplementary Table S5) was isolated from Level 5/6/7 (flowering stage) based on the two TO-GCNs (Figure 8 and Figure S1). At Level 5, ABF2 (ABA SIGNALING FACTORS), ARF (AUXIN RESPONSE FACTOR), and AP2/ERF (APETALA2/ETHYLENE RESPONSE ELEMENT BINDING FACTOR) transcription factors are co-expressed with MYB transcription factors, while the MYB gene is co-expressed with MeMADS12 at Level 6 (Figure 9). On the other hand, the MYB transcription factor is indirectly connected with MeMADS12 through VPS (VACUOLAR PROTEIN SORTING) (Figure 9). ABA is synthesized in various organelles such as plastids, the endoplasmic reticulum, and vacuoles, and ABA signal transduction and termination can be regulated through the vacuolar sorting pathway. Therefore, MeMADS12 is perhaps involved in ABA signaling pathways [43,44,45,46].
At Level 6, MeMADS12 is co-expressed with PP2C1 (PROTEIN PHOSPHATASE 2C), and MeMADS13 and MeMADS30 are co-expressed with PP2C2. At the same time, they are co-expressed with CDPK (CALCIUM-DEPENDENT PROTEIN KINASES) (Figure 9). At Level 7, MeMADS32 is co-expressed with upstream MeMADS30 and MeMADS13, and MeMADS22 is co-expressed with MeMADS32 and MeMADS72, respectively (Figure 9). Meanwhile, CDPK may be a signaling factor for the expression of MeMADS in the next level (Figure 9). It is worth noting that HY5 (LONG HYPOCOTYL 5) [47] is co-expressed with MeMADS72 and MeMADS32 at Level 7, indicating the involvement of light signals in flowering regulation (Figure 9). At Level 6 and Level 7, the NINJA (NOVEL INTERACTOR OF JAZ) protein [48] is involved in the co-expression network, suggesting that the JA signaling pathway is also involved in regulation (Figure 9).
In summary, during the drought-induced flowering stage, MeMADS transcription factors participate in the signaling pathways of ABA, auxin, ethylene, and JA. The expression dynamics of hormone biosynthesis genes may affect the expression of MeMADS, which further acts on downstream flowering genes and activates other subsequent plant responses.
In our study, MeMADS12 was found to be induced by drought. In the TO-GCN constructed from the transcriptome data of cassava during its flowering stage, the hierarchical regulatory network shows that MeMADS12 is a key gene linking the upper and lower regulatory networks. On the other hand, MeMADS12 belongs to the FUL group, and genes of this group have been proven to control flowering time and the formation of inflorescence structure in plants such as A. thaliana, Solanum lycopersicum, and Chrysanthemum morifolium [49,50,51,52]. Therefore, MeMADS12 might be a key gene regulating cassava flowering under drought conditions. Hence, we chose MeMADS12 for further analysis.

3.9. Vector Construction and Subcellular Localization of MeMADS12

Previous predictions indicate that MeMADS12 is localized in the nucleus. However, some studies have shown that MADS-box genes can also localize in the cytoplasm [53]. The KRR-4-KK motif, located at positions 22–30 of the MADS-box domain, is important in translocating MADS-box proteins into the nucleus [54]. This nuclear localization sequence (NLS) is present in all FUL proteins, including MeMADS12 (Supplementary Figure S2). To verify the localization of the protein expressed by the MeMADS12 as an indicator of function, a fusion expression vector of pCAMBIA1300-MeMADS12-GFP was constructed (Figure 10), and transient expression was mediated by Agrobacterium tumefaciens in tobacco leaves. The empty vector pCAMBIA1300 was used as the control. After 3 days of co-culturing, laser confocal microscopy showed that the fluorescence signal in cells transfected with the control vector was observed in the cell membrane, cytoplasm, and nucleus (Figure 10). The fluorescence signal corresponding to pCAMBIA1300-MeMADS12-GFP in transfected tobacco leaf cells could be observed in the nucleus (Figure 10). This is consistent with the forecast above.

3.10. Relative Expression Levels of MeMADS12 via RT-qPCR Analysis

The promoter region of MeMADS12 contains two binding sites related to drought response (MBS) and one site involved in abscisic acid responsiveness (Figure 5, Supplementary Table S4), suggesting that MeMADS12 may be involved in the ABA response pathway under drought induction. To determine the MeMADS12 in response to drought stress and ABA treatment, RT-qPCR analysis was used to estimate the expression of MeMADS12. For this experiment, the leaves and roots of two cassava cultivars were obtained after the two treatments. After drought and ABA treatments, MeMADS12 was upregulated in the leaf (Figure 11). For roots, the expression of MeMADS12 decreased in the Arg7 cultivar under drought stress. On the contrary, the expression of MeMADS12 was upregulated in the SC124 cultivar under drought stress. MeMADS12 was upregulated in two cultivars under ABA treatment. Different drought-tolerant varieties of cassava had different mechanisms to respond to drought. As a result, MeMADS12 was involved in the drought stress response as well as the ABA response.

4. Discussion

4.1. Identification and Evolutionary Analysis of Cassava MADS-Box Gene Family

In this study, 86 MADS-box genes in the cassava genome were identified and analyzed using bioinformatics methods. The phylogenetic analysis (Figure 4a) revealed that type I MADS-box genes in cassava can be classified into three subfamilies, namely, Mα, Mβ, and Mγ. Notably, the phylogenetic trees of the Mβ and Mγ subfamilies showed distinct differences compared to those of other subfamilies. Within these two subfamilies, homologous genes of different subfamilies were clustered together, followed by homologous genes of the same subfamilies aggregating into a group. This pattern of phylogenetic evolution suggested that gene duplications occurred independently within the two subfamilies, leading to the generation of numerous homologous genes after the divergence of these two subfamilies. Some research suggested that the presence of multiple introns represents a more primitive state of genes [55]. Therefore, type II MADS-box genes might represent a more ancestral form of MADS-box genes, whereas type I MADS-box genes emerged later in evolutionary history by comparison. In Arabidopsis and rice, the MIKC* subfamily genes are also referred to as type Mδ genes, and some researchers classify them as a subtype of type II genes [14,56]. However, in our study, MIKC* subfamily genes exhibit structural similarities to both type I and type II genes, and in terms of phylogenetic analysis, they are found to be closer to type I genes. Consequently, MIKC* subfamily genes are likely transitional forms in the evolution of MADS-box genes, thereby possessing characteristics of both type I and type II genes. The evolution of MADS-box genes may have proceeded as follows: sequence changes in type II genes led to the loss of the K domain, while their intron count remained unchanged, giving rise to the MIKC* subfamily. Subsequently, a reduction in the number of introns and the fusion of exons in MIKC* genes resulted in the emergence of the other three type I gene subfamilies (Mα, Mβ, and Mγ). Conserved motif analyses further substantiate this hypothesis. The K domain is composed of motif 2 and motif 5 in our study. Among the eight MIKC* subfamily genes in cassava, MeMADS80 and MeMADS16 notably retain motif 2. This result suggested that, during evolution, substantial sequence changes led to an incomplete K domain, thereby giving rise to the MIKC* subfamily genes. On the other hand, although MeMADS43 and MeMADS46 in the Mβ family still possess motif 2, their reduced number of introns implies that exon fusion events might have contributed to the gene differentiation within this subfamily. The loss of introns appears to be a prominent feature in the evolutionary trajectory of MADS-box genes, and it could be one of the factors contributing to the functional diversity observed in the cassava MADS-box gene family.

4.2. MADS-Box Genes’ Regulatory Mechanisms for Flowering in Response to Drought Stress

Indeed, flowering in cassava is crucial for the production of seeds used in plant breeding. Typically, cassava breeding relies heavily on vegetative propagation methods such as stem cuttings due to the plant’s low seed set and often poor germination rate. However, successful flowering and seed production are essential for genetic diversity enhancement and the development of new cultivars through hybridization and selection. Flowering allows for the creation of crosses between different genetic backgrounds, enabling breeders to introduce desirable traits, such as drought tolerance or disease resistance, into new varieties. Therefore, understanding the mechanisms that regulate flowering in cassava and promoting its natural reproductive process can significantly contribute to the advancement of cassava cultivation worldwide. In this study, a functional analysis of cis-regulatory elements in the promoter region of MeMADS genes was conducted, revealing the presence of an abscisic-acid-responsive element (ABRE) and two MYB transcription factor binding sites (MBS, a drought-stress-responsive element) in MeMADS12 (Supplementary Table S4). This result suggested that drought stress and ABA could induce the expression of MeMADS12. RNA-Seq data and RT-qPCR analysis further supported this finding by demonstrating that MeMADS12 was induced under drought treatment. In TO-GCN, MeMADS12 was found to be co-expressed with an MYB gene, implying an interaction between these two genes in regulating flowering under drought stress in cassava. The MYB gene co-expressed with MeMADS12 was annotated as Myb-related protein 306-like (MYB31), and the study of its function was focused on metabolite synthesis regulation [57,58,59]. However, in our study, the MYB gene was observed to be co-expressed with multiple hormone signaling factors, suggesting a broader role beyond metabolism regulation. Collectively, these findings underscore the need for further investigation into the specific functions and mechanisms of both the MYB gene and MeMADS12, particularly their potential interplay in mediating stress-responsive flowering pathways in cassava.
ABA plays a crucial role in the response to drought stress. Depending on their response to ABA, drought-stress-responsive genes can broadly be categorized into two types, namely, those that are dependent on ABA and those that are independent of ABA [60]. The ABA-dependent signaling system is described as mediating stress responses by activating at least two classes of regulatory factors [61], including the AREB/ABF transcription factors and the MYC/MYB transcription factors [62,63]. Our analysis of the subnetwork extracted from the TO-GCN suggested that MeMADS12 was involved in mediating stress responses via ABA-dependent pathways, and it might also rely on interactions with other plant hormones. On the other hand, MeMADS13 and MeMADS30 showed co-expression with PP2Cs [64,65,66] involved in the ABA signaling pathway and CDPKs [67,68,69,70] implicated in stress signaling, respectively. Notably, MeMADS30 also co-expresses with NINJA, which is part of the jasmonic acid signaling pathway. These results imply that different members of the MeMADS gene family might occupy distinct positions in the signaling cascades against abiotic stresses, sequentially transmitting stress signals and functioning as transcription factors to modulate the activity of downstream genes. Ultimately, this leads to the translation of proteins that confer resistance to abiotic stresses, thereby executing their biological functions.

5. Conclusions

In this study, we identified a total of 86 MADS-box genes with complete domains in the cassava genome, designated as MeMADS01 to MeMADS86. Through bioinformatic analyses, we investigated the basic physicochemical properties, conserved motifs, chromosomal locations, and phylogenetic relationships of the cassava MADS-box genes. The MADS-box gene family of cassava exhibited conservation, as well as species-specific characteristics, with intron loss being a predominant mode of evolution for the MADS-box genes. The expression pattern variations in the MeMADS genes across different tissues offer insights into their potential functions. TO-GCN, transcriptome data, and RT-qPCR analysis suggested the responsiveness of the MADS-box genes to drought stress. Meanwhile, MeMADS12 might be involved in regulating flowering under drought conditions via an ABA-dependent pathway. These findings provide valuable resources for a deeper understanding of the biological roles of the MADS-box genes in cassava.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/horticulturae10101073/s1: Supplementary Figure S1. TO-GCN with 7 levels constructed from TF genes. Supplementary Figure S2: Amino acid sequence alignment of FUL subgene family in cassava. Supplementary Table S1. The primer was used in experiments. Supplementary Table S2. List of MADS-box genes in 11 species and the genome size of 11 species. Supplementary Table S3. Physicochemical properties and subcellular localization analysis of cassava MADS-box genes. Supplementary Table S4. Results of PlantCARE for MeMADS genes in cassava. Supplementary Table S5. PCC value of subnetwork from Level 5/6/7. Supplementary Table S6. Annotation of genes from subnetwork.

Author Contributions

Conceptualization, Q.Z., Y.L., D.M. and M.X.; data curation, Q.Z., Y.L. and Q.L.; formal analysis, X.L.; investigation, S.G., S.S. and Z.S.; methodology, Q.Z., Y.L., Z.S. and D.M.; project administration, S.G. and W.Y.; resources, Q.L., Y.Z., S.S. and B.C.; software, Q.Z.; supervision, S.G., K.L. and W.Y.; validation, Q.Z.; visualization, Q.Z. and M.X.; writing—original draft, Q.Z.; writing—review and editing, Y.L., B.C., K.L. and W.Y. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Natural Science Foundation of China (32160398) and Yunnan Fundamental Research Projects (202301BD070001-221).

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Conflicts of Interest

Author Kang Li was employed by the company Changning County Lan Hui Agricultural Development Company Limited. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Figure 1. Phylogenetic tree and MADS-box gene numbers of M. esculenta and ten other species in the context of angiosperms.
Figure 1. Phylogenetic tree and MADS-box gene numbers of M. esculenta and ten other species in the context of angiosperms.
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Figure 2. Maximum likelihood phylogenetic tree constructed using eleven species of MADS-box genes. The MADS-box genes of different species are represented by different colored shapes.
Figure 2. Maximum likelihood phylogenetic tree constructed using eleven species of MADS-box genes. The MADS-box genes of different species are represented by different colored shapes.
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Figure 3. Chromosome location of MADS-box genes in M. esculenta. The scale on the left is in metabases (Mb). The colored lines within each chromosome represent gene density: the redder the color, the higher the density, and the bluer the color, the lower the density.
Figure 3. Chromosome location of MADS-box genes in M. esculenta. The scale on the left is in metabases (Mb). The colored lines within each chromosome represent gene density: the redder the color, the higher the density, and the bluer the color, the lower the density.
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Figure 4. Analysis of structure of M. esculenta MADS-box genes family. (a) Phylogenetic tree of M. esculenta MADS-box genes. (b) Conserved motif analysis. (c) Conserved domain analysis. (d) Structure of MADS-box genes in M. esculenta.
Figure 4. Analysis of structure of M. esculenta MADS-box genes family. (a) Phylogenetic tree of M. esculenta MADS-box genes. (b) Conserved motif analysis. (c) Conserved domain analysis. (d) Structure of MADS-box genes in M. esculenta.
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Figure 5. Analysis of cis-acting elements of MADS-box gene promoters in M. esculenta. The number in the box represents the number of cis-acting elements. The shade of the color block depends on the number of cis-acting elements.
Figure 5. Analysis of cis-acting elements of MADS-box gene promoters in M. esculenta. The number in the box represents the number of cis-acting elements. The shade of the color block depends on the number of cis-acting elements.
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Figure 6. Gene expression levels of MADS-box genes in M. esculenta in different tissues. The FPKM values of MADS-box genes are taken as the logarithm with base 2 for standardization. The patches of different colors indicate the expression levels of MADS-box genes in different tissues: from blue to red represents a gradual increase in value. The connecting lines on the left and top represent cluster analysis. FEC: friable embryogenic callus; OES: somatic organized embryogenic structure; RAM: root apical meristem; SAM: shoot apical meristem.
Figure 6. Gene expression levels of MADS-box genes in M. esculenta in different tissues. The FPKM values of MADS-box genes are taken as the logarithm with base 2 for standardization. The patches of different colors indicate the expression levels of MADS-box genes in different tissues: from blue to red represents a gradual increase in value. The connecting lines on the left and top represent cluster analysis. FEC: friable embryogenic callus; OES: somatic organized embryogenic structure; RAM: root apical meristem; SAM: shoot apical meristem.
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Figure 7. Gene expression levels of MADS-box genes in M. esculenta leaf and root under drought treatment. The FPKM values of MADS-box genes are taken as the logarithm with base 2 for standardization. The patches of different colors indicate the expression levels of MADS-box genes in different tissues: from blue to red represents a gradual increase in value. The connecting lines on the left represent cluster analysis. FEL_0h/ FEL_3h/ FEL_24h: full expanded leaf was collected after drought treatment for 0 h, 3 h, and 24 h, respectively; RT_0h/ RT_3h/ RT_24h: root was collected after drought treatment for 0 h, 3 h, and 24 h, respectively.
Figure 7. Gene expression levels of MADS-box genes in M. esculenta leaf and root under drought treatment. The FPKM values of MADS-box genes are taken as the logarithm with base 2 for standardization. The patches of different colors indicate the expression levels of MADS-box genes in different tissues: from blue to red represents a gradual increase in value. The connecting lines on the left represent cluster analysis. FEL_0h/ FEL_3h/ FEL_24h: full expanded leaf was collected after drought treatment for 0 h, 3 h, and 24 h, respectively; RT_0h/ RT_3h/ RT_24h: root was collected after drought treatment for 0 h, 3 h, and 24 h, respectively.
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Figure 8. Time-ordered gene co-expression network (TO-GCN) of the flowering cassava. The red, green, and blue colors represent MADS-box genes, TF, and non-TF genes, respectively. The data on each level represent the sampling time.
Figure 8. Time-ordered gene co-expression network (TO-GCN) of the flowering cassava. The red, green, and blue colors represent MADS-box genes, TF, and non-TF genes, respectively. The data on each level represent the sampling time.
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Figure 9. Subnetwork for MADS-box genes and multiple signaling-pathway-related genes. Red indicates the MeMADS genes and the orange-yellow indicates other transcription factors or proteins.
Figure 9. Subnetwork for MADS-box genes and multiple signaling-pathway-related genes. Red indicates the MeMADS genes and the orange-yellow indicates other transcription factors or proteins.
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Figure 10. Subcellular localization analysis of the MeMADS12 protein in tobacco leaf cells. The fluorescence signal is detected in transfected cells. From left to right, the picture is of a bright field, a GFP field, and a merged field with light and shade. The scale is 20 µm.
Figure 10. Subcellular localization analysis of the MeMADS12 protein in tobacco leaf cells. The fluorescence signal is detected in transfected cells. From left to right, the picture is of a bright field, a GFP field, and a merged field with light and shade. The scale is 20 µm.
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Figure 11. Relative expression levels of MeMADS12 of leaf and under drought stress and ABA treatment in cassava. (a) Relative expression levels of MeMADS12 of leaf under drought stress; (b) relative expression levels of MeMADS12 of leaf under ABA treatment; (c) relative expression levels of MeMADS12 of root under drought stress; (d) relative expression levels of MeMADS12 of root under ABA treatment. **: significant correlation at the 0.01 probability level (p < 0.01). Error bars indicate the SD of three independent biological and technical replicates.
Figure 11. Relative expression levels of MeMADS12 of leaf and under drought stress and ABA treatment in cassava. (a) Relative expression levels of MeMADS12 of leaf under drought stress; (b) relative expression levels of MeMADS12 of leaf under ABA treatment; (c) relative expression levels of MeMADS12 of root under drought stress; (d) relative expression levels of MeMADS12 of root under ABA treatment. **: significant correlation at the 0.01 probability level (p < 0.01). Error bars indicate the SD of three independent biological and technical replicates.
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Table 1. Analysis of evolutionary selection pressure on co-linear members of MADS-box genes in M. esculenta.
Table 1. Analysis of evolutionary selection pressure on co-linear members of MADS-box genes in M. esculenta.
Seq_1Seq_2KaKsKa_KsDuplication Type
MeMADS1MeMADS250.03320.27810.1195WGD or Segmental
MeMADS2MeMADS260.09220.28980.3181WGD or Segmental
MeMADS3MeMADS240.06660.36000.1851WGD or Segmental
MeMADS4MeMADS110.03050.31270.0976WGD or Segmental
MeMADS5MeMADS120.09210.37740.2441WGD or Segmental
MeMADS6MeMADS130.07300.48250.1513WGD or Segmental
MeMADS7MeMADS140.06380.30220.2111WGD or Segmental
MeMADS9MeMADS230.06400.34850.1835WGD or Segmental
MeMADS15MeMADS820.10660.49070.2173WGD or Segmental
MeMADS17MeMADS830.07270.30830.2359WGD or Segmental
MeMADS18MeMADS860.10870.32090.3387WGD or Segmental
MeMADS32MeMADS720.07190.35760.2010WGD or Segmental
MeMADS33MeMADS710.07000.21970.3186WGD or Segmental
MeMADS39MeMADS480.05640.25750.2191WGD or Segmental
MeMADS58MeMADS620.02720.26050.1042WGD or Segmental
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MDPI and ACS Style

Zhang, Q.; Li, Y.; Geng, S.; Liu, Q.; Zhou, Y.; Shen, S.; Shen, Z.; Ma, D.; Xiao, M.; Luo, X.; et al. Genome-Wide Identification and Expression Analysis of the MADS-Box Gene Family in Cassava (Manihot esculenta). Horticulturae 2024, 10, 1073. https://doi.org/10.3390/horticulturae10101073

AMA Style

Zhang Q, Li Y, Geng S, Liu Q, Zhou Y, Shen S, Shen Z, Ma D, Xiao M, Luo X, et al. Genome-Wide Identification and Expression Analysis of the MADS-Box Gene Family in Cassava (Manihot esculenta). Horticulturae. 2024; 10(10):1073. https://doi.org/10.3390/horticulturae10101073

Chicago/Turabian Style

Zhang, Qin, Yanan Li, Sha Geng, Qian Liu, Yingchun Zhou, Shaobin Shen, Zhengsong Shen, Dongxiao Ma, Mingkun Xiao, Xin Luo, and et al. 2024. "Genome-Wide Identification and Expression Analysis of the MADS-Box Gene Family in Cassava (Manihot esculenta)" Horticulturae 10, no. 10: 1073. https://doi.org/10.3390/horticulturae10101073

APA Style

Zhang, Q., Li, Y., Geng, S., Liu, Q., Zhou, Y., Shen, S., Shen, Z., Ma, D., Xiao, M., Luo, X., Che, B., Li, K., & Yan, W. (2024). Genome-Wide Identification and Expression Analysis of the MADS-Box Gene Family in Cassava (Manihot esculenta). Horticulturae, 10(10), 1073. https://doi.org/10.3390/horticulturae10101073

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