Skip to main content
NCBI home page
Physiology and Molecular Biology of Plants logoLink to Physiology and Molecular Biology of Plants
. 2022 Aug 22;28(7):1359–1374. doi: 10.1007/s12298-022-01216-1

Identification and expression analysis of the MADS-box genes of Kentucky bluegrass during inflorescence development

Jinqing Zhang 1, Huiling Ma 1,✉
PMCID: PMC9424482  PMID: 36051235

Abstract

MADS-box genes play vital roles in multiple biological processes of plants growth and development, especially inflorescence development. In the present study, a comprehensive investigation into the identification and classification of MADS-box genes in Kentucky bluegrass (Poa pratensis) has not been reported. Here, based on the transcriptome of inflorescence, we identified 44 PpMADS-box genes, and gave an overview of the physicochemical properties, phylogeny, protein structures, and potential functions of the proteins encoded by these genes through various bioinformatics software for the first time. Analysis of physicochemical properties revealed that most PpMADS-box were alkaline proteins and possessed similar conserved motifs. Additionally, it was demonstrated that 33 PpMADS-box proteins without signal peptide, leading peptide, transmembrane structure and located in the nucleus were not transported or secreted, so directly played transcriptional regulatory roles in the nucleus. Then, peptide sequences BLAST search and analysis of phylogenetic relationships with MADS-box proteins of P. pratensis, Arabidopsis thaliana, and Oryza sativa were performed. It was found that 44 PpMADS-box proteins were separated into 33 MIKC-type (3 BS, 1 AGL17, 8 AP3/P2, 3 AP1, 5 SEP, 6 SOC and 7 AG genes, respectvely) and 11 type I-type, which include 7 Mγ and 4 Mα. Furthermore, the relative expression levels of the selected 12 genes (MADS3, 15, 16, 17, 18, 20, 24, 27, 30, 36, 38 and 40) at the booting stage, pre-anthesis, anthesis, post-anthesis, and seed filling stage of inflorescences, as well as leaves and roots of the corresponding stages of inflorescences were analyzed, showing that most PpMADS-box genes were highly expressed mainly in young leaves and later inflorescences, and had complex patters in roots. Morever, except for PpMADS30 being highly expressed in the leaves, others were significantly highly expressed in inflorescence and/ or roots, demonstrating PpMADS-box genes also regulate leaves and roots development in plant. This study provides valuable insights into the MADS-box family genes in Kentucky bluegrass and its potential functional characteristics, expression pattern, and evolution in floral organogenesis and even reproduction development. @media print { .ms-editor-squiggler { display:none !important; } } .ms-editor-squiggler { all: initial; display: block !important; height: 0px !important; width: 0px !important; }

Supplementary Information

The online version contains supplementary material available at 10.1007/s12298-022-01216-1.

Keywords: Poa pratensis, MADS-box genes, Flower development, Developmental stages, Various organs, Expression pattern

Introduction

Flower is an important organ for reproduction in angiosperms and one of the most diverse organs in plants. Its development and structure are a highly complex involving many factors and their interactions. Meanwhile, it is critical for the study of the evolution and classification of plants (Cho et al. 2017). The specific process of flower development is that the stem apex is transformed into inflorescence meristem, which is then differentiated into various organs. The difference in monocotyledons, especially Gramineae, is that the inflorescence meristem first produces spikelet meristem, and then the spikelet meristem differentiates into floret meristem (Chandler 2011). The initiation and differentiation of floral organs are a joint mechanism of transcriptional regulation and interaction of multiple genes and response pathways. Under this mechanism, the MADS-box genes play key regulatory roles. Different temporal and spatial expression of MADS-box will specialize into different morphological structures, which will determine phenotypic differences (Chopy et al. 2021). Mean while, the response of the downstream target genes of the MADS-box also determines the diversification of floral organs (Theissen 2001). Additionally, the MADS-box genes encode transcription factors, which also play essential roles in the formation of floral structures. The main function of MADS-box genes is to control the process and switch of flower development by coordinating the expression changes of characteristic genes in various meristems, and to establish new development programs while inhibiting previous development programs (Sri et al. 2020; Ma et al. 2021). In addition to regulating the formation and differentiation of floral organs, flowering time and fruit development and maturation of flowering plants (Zhao et al. 2021a; Zhou et al. 2021), the MADS-box genes are also expressed in other organs (Sharma et al. 2020; Zhao et al. 2021b), such as the expression of MdDAM1 is limited to buds and control the growth arrest and bud dormancy (Moser et al. 2020). Moreover, MADS-box genes are involved in regulation of root growth and nodule formation, phyllodes transformation, vernalization, apical meristems differentiation, ovules development, photosynthesis and nutritional metabolism (Cullerne et al. 2021; Kumar et al. 2021; Sun et al. 2021).

The regulatory function of MADS-box proteins in the flower development can be summarized as an ABCDE genetic model according to organ determinants. A + E, A + B + E, B + C + E, C + E and C + D + E gene combination regulates the development of sepals, petals, stamens, carpels, and ovules, respectively (Theißen and Gramzow 2016; Theißen et al. 2016; Alhindi and Al-Abdallat 2021; Chen et al. 2021). Based on phylogenetic analysis, MADS-box genes can be classified into Type I and Type II lineages (Singh et al. 2021), in which Type I-type includes Mα, Mβ and Mγ subfamily (Alvarez-Buylla et al. 2000), and Type II-type is also called MIKC gene, which can be classified into MIKCC and MIKC* two subtypes (Kwantes et al. 2012; Qu et al. 2019). MIKCC is subdivided into 14 sub-families: AG/STK, AGL6, OsMADS32, AP1 (SQUA), AGL17, Bsiste (BS/GGM13), SEP1, FLC, SEP3, AGL12, SVP (StMADS11), TM3/SOC1, AP3 (DEF), PI (GLO) (Chen et al. 2017; Schilling et al. 2020), of which FLC, SOC1, SVP, AP1-FUL belong to class A genes, PI and AP3 belong to class B genes, BS and AG belong to class C genes, and SEP is the main class E gene (Gramzow and Theißen 2015).

The famous ABC model is based on the study of homeotic mutants that control flower development in model plants (Bowman et al. 1991; Chen et al. 2021), and then developed into the classic ABCDE model (Wen et al. 2019). Due to the floral organs are conservative in development and function, various researchers have proposed that the floret structure of monocots and dicots have the corresponding homologous relationship between the inner and outer lemma and the sepal, and between the paddle and the petal (Kong et al. 2019). Meanwhile, the classic ABCDE model of flower organ recognition of angiosperm is a conservative mechanism, and MADS-box genes are also conserved (Zhang et al. 2021c). Therefore, the ABCDE model can be at least partially applicable to the floret development of grasses (Kater et al. 2006). For example, SiMADS34 is essential for regulating inforescence architecture and grain yield in millet (Setaria italica) (Hussin et al. 2021). Kentucky bluegrass (Poa pratensis) can be used as an excellent pasture, and also a common cold-season turfgrasses. It has the ecological value of improving soil structure and fertility (Zhang et al. 2021a). So far, researches on MADS-box genes on floret development in Gramineous plants are concentrated on food crops such as wheat (Triticum aestivum) (Dixon et al. 2018) and rice (Oryza sativa) (Wu et al. 2018), with extremely little attention to the floret development of Kentucky bluegrass. Therefore, the wild Kentucky bluegrass germplasm materials is used, based on high-throughput sequencing data, and used bioinformatics methods to identify the MADS-box genes in Kentucky bluegrass. Their physicochemical properties, functional domains, guide peptides, signal peptides, transmembrane structure, subcellular localization, protein structure, and hydrophobicity were predicted and analyzed. Additionally, a phylogenetic tree was constructed to further study the evolution and classification of PpMADS-box genes. Furthermore, we selected several PpMADS-box genes to analyze their expression patterns in different organs at various stages to provide a theoretical basis for any further study on MADS-box genes in grasses.

Materials and methods

Experimental materials

The experimental materials were wild germplasm materials of Kentucky bluegrass collected in Longnan, Gansu Province of China. The materials were planted in the lawn training base of Gansu Agricultural University with single planting method. Starting from the booting stage, the inflorescences (I1-I5, I1: the booting stage; I2: pre-anthesis; I3: anthesis; I4: post-anthesis; I5: seed filling stage), as well as leaves (L1-L5) and roots (R1-R5) of the corresponding stages of inflorescences were collected, respectively. All samples were frozen in liquid nitrogen and stored at − 80 °C to analyze the expression patterns of several MADS-box genes of Kentucky bluegrass at different developmental stages in various organs. Three biological repeats were set at each sampling stage, and each sample was obtained from a mixture of at least 5 plants.

Identification, screening and classification of MADS-box genes in Kentucky bluegrass

The detailed information of the Kentucky bluegrass inflorescence transcriptome was derived from previously published literature, including two wild germplasm materials of Kentucky bluegrass from Gannan (GN) and Longnan (LN), Gansu Province of China at premeiosis (PreM), meiosis (M), postmeiosis (PostM), and anthesis (A) four developmental stages, among which LN with high apomictic rate and GN was opposite (Zhang and Ma 2022). The local transcriptome database was constructed using Bioedit software, and the OsMADS-box protein sequences of rice were used as the request sequences, using tblastn search for PpMADS-box (expectation value: 1 e-20), and corresponding proteins sequences were extracted by TBtools software (Chen et al. 2020). All protein sequences were used in CDD (conserved domain database) (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi), SMART (https://smart.embl.de/) and Pfam (https://pfam.xfam.org/) database to predicts the conserved domains. PpMADS-box domains were obtained after removing the repetitive sequences and redundant transcripts. PpMADS-box genes were classified according to Parenicová et al. (2003) and Arora et al. (2007). The MADS-box protein sequences of A. thaliana and O. sativa were downloaded from the Ensembl Plants (http://plants.ensembl.org/index.html) database.

Analysis of physicochemical properties and motif prediction of PpMADS-box proteins

Preliminary prediction of physicochemical properties of PpMADS-box proteins, including the number of amino acids, molecular weight, theoretical isoelectric point (pI), instability index, aliphatic index, and grand average of hydropathicity (GRAVY) were performed by Prot Param (http://web.expasy.org/prot param/) tool available on ExPASy online website. The subcellular localization of each PpMADS-box protein was predicted by using the online software Prot Comp 9.0 (http://linux.softberry.com). Motif types and arrangements in the PpMADS-box proteins were analyzed by online software MEME (http://meme-suite.org/), and visualization of the motif characteristics was performed using TBtools software. The settings were restricted to 10 motifs using MEME find, and Hidden Markov model (HMM) profiles for the MADS-box structural domain (PF00319, PF01486) were downloaded from the Pfam database.

Analysis of signal peptides, leading peptides and transmembrane structures of PpMADS-box proteins

Signal peptides and transmembrane structures of PpMADS-box proteins were analyzed using SignalP 5.0 (http://www.cbs.dtu.dk/services/SignalP-5.0) and TMHMM 2.0 (http://www.cbs.dtu.dk/services/TMHMM2.0) online software, respectively. And Target P-2.0 (http://www.cbs.dtu.dk/services/Target P-2.0) was used for leading peptides prediction.

Secondary and tertiary structural analysis of PpMADS-box proteins

Secondary structures of PpMADS-box proteins were predicted by using SOPMA (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.plpage=npsa_sopma.The html) online software, and we obtained secondary structures in different conformations such as α-helix, extended chain, β-turned angle and random curl. Tertiary structure of PpMADS-box proteins were predicted by SWISS-MODEL (http: //swissmodel.expasy.org/interactive) software.

Analysis of conserved motif and phylogenetic tree of the PpMADS-box proteins

Using the Clustal W program of the MEGA 7.0 software, multiple sequence comparisons were performed for Kentucky bluegrass and some functionally well-defined A. thaliana and O. sativa MADS-box proteins. Phylogenetic trees were constructed using the maximum-neighbor-Joining method (Neighbor-Joining), and estimated using the Jones-Thornton-Taylor (JTT) model algorithm. Bootstrap analysis was performed, and the repeat spreading value was set to 1000. Other parameters were set at default. The phylogenetic tree was visualized and embellished by the online software Evolview (https://www.evolgenius.info/evolview).

Expression pattern analysis of selected several PpMADS-box genes

The expression of each gene fragment in the Kentucky bluegrass transcriptome sequencing library was calculated using the FPKM (fragments per kilobase million) (Trapnell et al. 2010). After normalizing the data, TBtools software was used to draw heatmap of the expression of the PpMADS-box genes in GN and LN at PreM, M, PostM and A four stages (Zhang and Ma 2022).

12 PpMADS-box genes were selected for quantitative real-time PCR. The Primer-BLAST of NCBI (http://www.ncbi.nlm.nih.gov/tools/primer-blast) was used to design the specific qRT-PCR primers, and the total RNA was extracted by RNasy Plant Mini Kit (Qiagen, Hilden, Germany) from 45 samples. The reverse transcription was done using Prime Script RT reagent Kit with a gDNA Eraser (Perfect Real Time) (TaKaRa, Japan) according to the manufacturer's instruction. The qRT-PCR was performed with a StepOnePlus™ Real-Time PCR System (ABI) using SYBR premix Ex Taq (Takara, Japan). The qRT-PCR was repeated in triplicate. The reaction volume was 20 μL and included 2 × SuperReal PreMix Plus 10 μL, ddH2O 3 μL, cDNA template 5 μL, and forward and reverse primer 2 μL. The reaction conditions included pre-denaturation at 95 °C for 15 min and 40 cycles of PCR amplification, including denaturation at 95 °C for 10 s and annealing at 58 °C for 30 s. Using Actin as the internal reference gene, the relative expression levels of the selected genes were calculated by 2−∆∆Ct (Yang 2012).

Data analysis

All data were subjected to analysis of the one-way ANOVA (P < 0.05) using SPSS version 19.0 (IBM Corp., Armonk, NY, USA) for Windows and GraphPad Prism 8.0.2. software was used for drawing.

Result

Identification and analysis of physicochemical properties of the PpMADS-box proteins

A total of 44 PpMADS-box genes were identified based on the transcriptome of Kentucky bluegrass inflorescence (Zhang and Ma 2022), which were named PpMADS1-PpMADS44 (Table 1, Table S1), and in which 33 genes belonged to MIKC-type and 11 to Type I-type. The accession numbers of all 44 PpMADS-box genes in the GenBank database at NCBI were obtained. Analyses of the physicochemical properties of PpMADS-box proteins found that the numbers of amino acids ranged from 44 to 372. The molecular weight ranged from 5111.33 to 39,387.53. The pIs were between 4.49 and 11.38, and the pIs of 10 PpMADS proteins were less than 7.5, which belonged to acidic proteins and the remaining 34 belonged to alkaline proteins. The instability indexes ranged from 25.60 to 74.80, and the instability indexes of 9 PpMADS proteins were less than 40 being stable proteins and the remaining 35 being unstable. The aliphatic indexes ranged from 64.27 to 105.25, and the aliphatic indexes of 42 PpMADS proteins were less than 100 being hydrophilic proteins, only PpMADS2 and PpMADS26 being hydrophobic proteins. By a further subdivision, only the GRAVY value of PpMADS37 was greater than 0.5 being a hydrophobic protein, and the GRAVY value of 19 PpMADS proteins were less than − 0.5 being hydrophilic. And the GRAVY of the remaining 24 proteins were between − 0.5 and 0.5, which were amphiphilic proteins. The prediction of subcellular localization showed that most of the PpMADS proteins were localized in the nucleus. In addition, PpMADS1 and PpMADS4 were localized in the mitochondria, PpMADS28 and PpMADS39 in the Golgi, and PpMADS43 in the plasma membrane.

Table 1.

Analysis of physicochemical properties and subcellular localization of PpMADS-box proteins

Gene ID Work name accession number Type The numbers of amino acid Molecular weight/kD pI Instability index Aliphatic index GRAVY Subcellular localization
Cluster-30586.0 PpMADS1 ON993403 Type Ι 170 18,814.73 9.43 38.67 97.59  − 0.226 Mitochondrial
Cluster-32012.0 PpMADS2 ON993431 MIKC 139 15,230.72 4.81 34.15 105.25 0.139 Nuclear
Cluster-33357.1 PpMADS3 ON993398 Type Ι 256 28,089.99 7.65 37.55 70.16  − 0.473 Nuclear
Cluster-36706.0 PpMADS4 ON993396 Type Ι 44 5111.33 11.07 71.73 64.32  − 0.793 Nuclear
Cluster-42777.0 PpMADS5 ON993411 MIKC 222 24,833.45 9.31 49.31 86.22  − 0.494 Nuclear
Cluster-49928.0 PpMADS6 ON993399 Type Ι 129 13,853.83 10.11 66.58 81.09  − 0.231 Nuclear
Cluster-51324.101355 PpMADS7 ON993392 Type Ι 355 39,387.53 10.62 64.84 77.21  − 0.458 Mitochondrial
Cluster-51324.107963 PpMADS8 ON993405 MIKC 147 16,467.84 10.51 58.85 74.35  − 0.656 Nuclear
Cluster-51324.107964 PpMADS9 ON993406 MIKC 112 11,928.58 6.89 42.79 91.52 0.017 Nuclear
Cluster-51324.107965 PpMADS10 ON993413 MIKC 119 13,563.18 9.89 59.68 74.54  − 0.808 Nuclear
Cluster-51324.107966 PpMADS11 ON993407 MIKC 108 11,754.53 8.64 44.91 92.13 0.042 Nuclear
Cluster-51324.119058 PpMADS12 ON993410 MIKC 103 11,645.40 10.23 64.24 71.94  − 0.564 Nuclear
Cluster-51324.150211 PpMADS13 ON993408 MIKC 164 17,660.98 9.84 62.69 64.27  − 0.667 Nuclear
Cluster-51324.155683 PpMADS14 ON993404 MIKC 103 11,694.72 9.58 48.16 94.76  − 0.112 Nuclear
Cluster-51324.156903 PpMADS15 ON993412 MIKC 146 16,742.11 9.79 43.00 81.44  − 0.738 Nuclear
Cluster-51324.157981 PpMADS16 ON993420 MIKC 74 8389.74 10.41 69.49 81.76  − 0.511 Nuclear
Cluster-51324.168915 PpMADS17 ON993417 MIKC 79 8982.47 10.35 39.19 90.00  − 0.347 Nuclear
Cluster-51324.170560 PpMADS18 ON993424 MIKC 132 15,292.24 9.44 51.05 95.83 0.008 Nuclear
Cluster-51324.186665 PpMADS19 ON993421 MIKC 212 23,360.77 9.42 50.95 75.57  − 0.504 Nuclear
Cluster-51324.186666 PpMADS20 ON993422 MIKC 194 21,669.78 9.43 48.82 71.44  − 0.55 Nuclear
Cluster-51324.188422 PpMADS21 ON993402 Type Ι 372 39,633.81 4.49 37.91 71.45  − 0.322 Nuclear
Cluster-51324.195031 PpMADS22 ON993393 Type Ι 209 23,670.39 9.59 51.64 84.93  − 0.566 Nuclear
Cluster-51324.209951 PpMADS23 ON993400 Type Ι 152 16,311.41 5.28 54.27 82.96  − 0.127 Nuclear
Cluster-51324.30204 PpMADS24 ON993414 MIKC 169 18,953.01 8.97 58.14 69.41  − 0.973 Nuclear
Cluster-51324.55798 PpMADS25 ON993426 MIKC 124 14,382.77 9.35 41.26 82.58  − 0.528 Nuclear
Cluster-51324.55799 PpMADS26 ON993425 MIKC 52 6006.31 10.12 34.91 105.00  − 0.25 Nuclear
Cluster-51324.59905 PpMADS27 ON993395 Type Ι 298 32,576.99 7.25 41.92 73.89  − 0.402 Nuclear
Cluster-51324.62676 PpMADS28 ON993430 MIKC 140 15,423.37 9.03 36.92 75.93 0.251 Golgi
Cluster-51324.70025 PpMADS29 ON993432 MIKC 136 15,272.03 10.01 50.65 86.18  − 0.425 Nuclear
Cluster-51324.73533 PpMADS30 ON993423 MIKC 268 30,779.05 9.31 65.18 74.66  − 0.841 Nuclear
Cluster-51324.79050 PpMADS31 ON993409 MIKC 92 9743.25 9.91 78.67 72.28  − 0.427 Nuclear
Cluster-51324.82899 PpMADS32 ON993435 MIKC 145 16,485.04 5.82 46.05 90.21  − 0.566 Nuclear
Cluster-51324.87676 PpMADS33 ON993429 MIKC 109 12,540.19 6.45 25.60 85.87  − 0.75 Nuclear
Cluster-51324.87686 PpMADS34 ON993433 MIKC 89 10,280.60 7.78 47.06 90.90  − 0.921 Nuclear
Cluster-51324.87690 PpMADS35 ON993434 MIKC 108 12,350.36 9.1 28.81 75.83  − 0.925 Nuclear
Cluster-51324.90311 PpMADS36 ON993415 MIKC 272 31,398.02 7.31 71.31 65.96  − 1.076 Nuclear
Cluster-51324.90312 PpMADS37 ON993428 MIKC 237 26,366.66 9.79 67.03 65.99 0.899 Nuclear
Cluster-51324.91461 PpMADS38 ON993419 MIKC 251 28,262.63 6.59 48.93 90.96  − 0.221 Nuclear
Cluster-51324.95134 PpMADS39 ON993416 MIKC 119 13,445.27 4.72 58.64 82.69  − 0.339 Golgi
Cluster-51324.95967 PpMADS40 ON993401 MIKC 110 12,564.40 8.26 45.44 82.45 0.095 Nuclear
Cluster-51324.96956 PpMADS41 ON993418 MIKC 121 13,283.19 9.94 48.22 76.61  − 0.346 Nuclear
Cluster-56886.0 PpMADS42 ON993397 Type Ι 260 28,446.63 9.53 50.80 72.00  − 0.55 Nuclear
Cluster-57814.2 PpMADS43 ON993427 MIKC 237 24,534.39 11.38 74.80 78.69  − 0.158 Plasma membrane
Cluster-65884.0 PpMADS44 ON993394 Type Ι 206 22,858.38 9.33 51.44 88.98  − 0.33 Nuclear
Open in a new tab

Analysis of the conserved motif structure of the PpMADS-box proteins

The Clustal W was used to perform multiple sequence comparisons of the PpMADS-box proteins to remove the non-conserved regions, and then motifs were analyzed to find the conserved structural domains (Fig. 1). It was found that the MIKC-type PpMADS-box proteins contained 41 amino acid residues, including seven extremely conserved amino acid residues, such as lysine residues (K) at the 15th and 23rd, arginine residue (R) at the 16th, glutamate residue (E) at the 26th, leucine residue (L) at the 30th, cysteine residue (C) at the 31st, and alanine residue (A) at the 33rd. Furthermore, there were many other conserved amino acid residues, such as the valine residue (V) at the 11th, the phenylalanine residues (F) at the 13th and 40th and so on. The conserved motif of Type I-type PpMADS-box has only 29 amino acid residues, of which the extremely conserved amino acid residues were K at the 9th and L at the 20th, and the other conserved amino acid residues included R at the 2nd, K at the 15th, L at the 23rd, and C at the 24th. Overall, PpMADS-box proteins were relatively conserved, especially for amino acid residues such as K, L, R, E, C, F. Comparing and analyzing the two types of proteins, it was found that the number of amino acids, were extremely conserved and conserved amino acid residues of the MIKC-type PpMADS-box proteins were far more than those of the Type I-type, indicating that the MIKC-type PpMADS-box proteins were more conserved than Type I-type.

Fig. 1.

Fig. 1

Open in a new tab

The structures of the most important and conserved motif of PpMADS-box proteins. Here, the motifs are motif1 from Supplementary Fig. S1, which are the most important and conserved motifs of PpMADS-box, so their structures can basically represent all PpMADS-box proteins. A: MIKC-type PpMADS-box proteins; B: Type I-type PpMADS-box proteins

Conserved motif analysis of PpMADS-box proteins

MEME was used to analyze the conserved motifs of PpMADS-box proteins, and 10 conserved motifs were obtained for each MIKC and Type I-type MADS-box protein, respectively (Fig. 2 and S1). The results showed that among 33 MIKC-type PpMADS-box, except for PpMADS9, PpMADS31, PpMADS32, and PpMADS34, the remaining 29 proteins contained motif1 or motif2. Most of the MIKC-type proteins contained motif1, motif2, or motif5, and PpMADS37 only contained motif1. Furthermore, PpMADS31 did not contain any motif, which may be due to the short length of the gene sequence. Among the 11 Type I-type PpMADS-box, except for PpMADS21 which only contained motif4, the other 10 proteins contained motif1.

Fig. 2.

Fig. 2

Open in a new tab

Motifs of PpMADS-box proteins, and phylogenetic tree build based on motifs. A: Motifs of MIKC-type; B: Motifs of Type I-type

Analysis of signal peptides, leading peptides, transmembrane and secondary structure of PpMADS-box proteins

The signal peptide of the PpMADS-box proteins were analyzed, and the results showed that only PpMADS2 and PpMADS11 have a signal peptide, respectively (Table 2, Fig. S2A, B), which were secreted proteins, and the remaining proteins were non-secreted proteins. The predictive analysis of the leading peptide showed only PpMADS6 had a mitochondrial transit peptide (Table 2, Fig. S2C), and PpMADS27 had a chloroplast transit peptide (Table 2, Fig. S2D). In addition, other remaining proteins did not contain any leading peptide. Transmembrane structural domain analysis revealed only PpMADS19 and PpMADS40 respectively had a transmembrane structural domain, and others did not have any transmembrane structure. The typical transmembrane structural domain of PpMADS19 located at the 59th–81st amino acids, with the 1st–58th amino acids located on the inner surface of the cell membrane and the 82nd–212nd amino acids on the outer surface of the cell membrane. The 1st–81st amino acids of PpMADS40 located on the inner surface of the cell membrane, and the 105th–110th amino acids located on the outer surface of the cell membrane. And the 82nd–104th amino acids formed a typical transmembrane helical region (Table 2, Fig. S2F).

Table 2.

Analysis of signal peptides, leading peptides, transmembrane and secondary structure of PpMADS-box proteins

name Signal peptide Mitochondrial Chloroplast Thylakoid luminal Transmembrane Secondary structure
transfer peptide transfer peptide transfer peptide structure α- helix extended chain β-turned angle random curl
PpMADS1 0.0008 0 0.0036 – – 0 100(58.82%) 27(15.88%) 13(7.65%) 30(17.65%)
PpMADS2 0.5136 1 0.0109 0.0028 0.0001 0 102(73.38%) 13(9.35%) 8(5.76%) 16(11.51%)
PpMADS3 0.0008 0 0.0001 – – 0 129(50.39%) 13(5.08%) 10(3.91%) 104(40.62%)
PpMADS4 0.0022 0 0.0003 – – 0 27(61.36%) 6(13.64%) 2(4.55%) 9(20.45%)
PpMADS5 0.0017 0 0.0026 0.0049 0.0604 0 118(53.15%) 23(10.36%) 14(6.31%) 67(30.18%)
PpMADS6 0.0068 0 0.5722 0.0595 0.0886 0 60(46.51%) 20(15.50%) 9(6.98%) 40(31.01%)
PpMADS7 0.0015 0 0 0.0001 0 0 116(32.68%) 48(13.52%) 27(7.61%) 164(46.20%)
PpMADS8 0.005 0 0.0156 – – 0 63(42.86%) 29(19.73%) 16(10.88%) 39(26.53%)
PpMADS9 0.062 0 0.0002 0.0051 0.0007 0 36(32.14%) 21(18.75%) 12(10.71%) 43(38.39%)
PpMADS10 0.0005 0 0 0 0 0 57(47.90%) 23(19.33%) 12(10.08%) 27(22.69%)
PpMADS11 0.581 1 0.0015 – – 0 39(36.11%) 25(23.15%) 5(4.63%) 39(36.11%)
PpMADS12 0.0021 0 0 – – 0 49(47.57%) 15(14.56%) 14(13.59%) 25(24.27%)
PpMADS13 0.0009 0 0.0006 – – 0 17(10.73%) 34(20.73%) 22(13.41%) 91(55.49%)
PpMADS14 0.0007 0 0.0087 0 0 0 57(55.34%) 17(16.50%) 9(8.74%) 20(19.42%)
PpMADS15 0.0002 0 0.0015 – – 0 90(64.64%) 20(13.70%) 6(4.11%) 30(20.55%)
PpMADS16 0.001 0 0.0029 0 0 0 30(40.54%) 16(21.62%) 10(13.51%) 18(24.32%)
PpMADS17 0.0011 0 0.0653 – – 0 25(31.65%) 24(30.38%) 11(13.92%) 19(24.05%)
PpMADS18 0.0007 0 0.0039 0 0.0001 0 57(43.18%) 34(25.76%) 10(7.58%) 31(23.48%)
PpMADS19 0.0018 0 0.0011 – – 1 106(50.00%) 31(14.62%) 16(7.55%) 59(27.83%)
PpMADS20 0.0011 0 0 0.0001 0.0435 0 97(50.00%) 28(14.43%) 13(6.70%) 56(28.87%)
PpMADS21 0.0361 0 0.0036 – – 0 87(23.39%) 62(16.67%) 19(5.11%) 204(54.84%)
PpMADS22 0.0016 0 0.0019 0 0 0 106(50.72%) 12(5.74%) 8(3.83%) 83(39.71%)
PpMADS23 0.0005 0 0.039 – – 0 83(54.61%) 20(13.16%) 12(7.89%) 37(24.34%)
PpMADS24 0.0014 0 0.0002 – – 0 83(49.11%) 19(11.24%) 12(7.10%) 55(32.54%)
PpMADS25 0.0004 0 0.0005 – – 0 71(57.26%) 21(16.94%) 9(7.26%) 23(18.55%)
PpMADS26 0.001 0 0.0002 0 0 0 25(48.08%) 12(23.08%) 10(19.23%) 5(9.62%)
PpMADS27 0.0082 0 0.0136 0.7292 0.016 0 117(39.26%) 28(9.40%) 7(2.35%) 146(48.99%)
PpMADS28 0.0016 0 0.0021 0.003 0.0007 0 12(8.57%) 54(38.57%) 18(12.86%) 56(40.00%)
PpMADS29 0.0009 0 0.0005 – – 0 55(40.44%) 30(22.06%) 12(8.82%) 39(28.68%)
PpMADS30 0.0008 0 0.0001 – – 0 155(57.84%) 24(8.96%) 10(3.73%) 79(29.48%)
PpMADS31 0.001 0 0 0 0 0 40(43.48%) 9(9.78%) 7(7.61%) 36(39.13%)
PpMADS32 0.0006 0 0.0001 – – 0 83(57.24%) 17(11.72%) 12(8.28%) 33(22.76%)
PpMADS33 0.002 0 0.0002 0 – 0 75(68.81%) 9(8.26%) 6(5.50%) 19(17.43%)
PpMADS34 0.0003 0 0 0 0 0 71(79.78%) 0(0.00%) 0(0.00%) 18(20.22%)
PpMADS35 0.0006 0 0.0126 – – 0 80(74.07%) 2(1.85%) 3(2.78%) 23(21.30%)
PpMADS36 0.001 0 0 0 0 0 142(52.21%) 25(9.19%) 8(2.94%) 97(35.66%)
PpMADS37 0.0007 0 0.0002 – 0 0 69(29.11%) 38(16.03%) 24(10.13%) 106(44.73%)
PpMADS38 0.0014 0 0.0501 – – 0 136(54.18%) 34(13.55%) 16(6.37%) 65(25.90%)
PpMADS39 0.0045 0 0.2593 0.0001 0 0 93(64.14%) 15(10.34%) 5(3.45%) 32(22.07%)
PpMADS40 0.0025 0 0.0003 – – 1 37(30.58%) 28(23.14%) 15(12.40%) 41(33.88%)
PpMADS41 0.0007 0 0.002 0.3676 0.0156 0 48(43.64%) 27(24.55%) 3(2.73%) 32(29.09%)
PpMADS42 0.0006 0 0 0.0001 0.0031 0 122(46.92%) 28(10.77%) 13(5.00%) 97(37.31%)
PpMADS43 0.0071 0 0.0001 0.2689 0.0011 0 48(20.25%) 32(13.50%) 15(6.33%) 142(59.92%)
PpMADS44 0.0009 0 0.0018 – – 0 128(62.14%) 10(4.85%) 6(2.91%) 62(30.10%)
Open in a new tab

Analysis of the secondary and tertiary structure of the PpMADS-box proteins

The results of secondary structure found the largest proportion of the secondary structure of the 10 MADS-box proteins was random curl, while the largest proportion of the secondary structure of the remaining 34 proteins was α-helical overall. The percentage of secondary structure composition of the most PpMADS-box proteins exhibited α-helix > random curl > extended chain > β-turned angle (Table 2). The tertiary structure showed significant differences due to the different proportions of secondary structure composition (Fig. 3A). Among them, the structure of Fig. 3a–e represents 9 proteins of PpMADS-box (PpMADS5, 6, 10, 17, 18, 23, 25, 36, 41), Fig. 3a–c represents 6 proteins of PpMADS-box (PpMADS3, 15, 22, 27, 30, 42), Fig. 3a–p (PpMADS24, 29 and 38) and 3A-u (PpMADS34, 35 and 39) each represent 3 proteins, and Fig. 3a–d (PpMADS4 and 26) and a–k (PpMADS13 and 19) each represent 2 proteins, respectively. And the remaining 19 PpMADS proteins each have their own unique spatial structure.

Fig. 3.

Fig. 3

Open in a new tab

Analysis of tertiary structures and expression pattern of PpMADS-box proteins. A: The tertiary structure of PpMADS-box proteins. a: MADS1; b: MADS2; c: MADS3, 15, 22, 27, 30, 42; d: MADS4, 26; e: PpMADS5, 6, 10, 17, 18, 23, 25, 36, 41; f: MADS7; g: MADS8; h: MADS9; i: MADS11; j: MADS12; k: MADS13, 19; l: MADS14; m: MADS16; n: MADS20; o: MADS21; p: MADS24, 29, 38; q: MADS28; r: MADS31; s: MADS32; t: MADS33; u: MADS34, 35, 39, v: MADS37; w: MADS40; x: MADS43; y: MADS44. B: Analysis of expression pattern of PpMADS-box genes. LPreM, LM, LPostM and LA represent inflorescence samples at premeiosis, meiosis, postmeiosis, and anthesis stage respectively in Longnan, which has high apomictic rate. And GPreM, GM, GPostM and GA represent these in Gannan, which has low apomictic rate

Phylogenetic tree analysis of MADS-box proteins from P. pratensis, A. thaliana, and O. sativa

The phylogenetic tree of the AtMADS-box proteins in A. thaliana, OsMADS-box proteins in rice, and PpMADS-box proteins in Kentucky bluegrass were constructed (Fig. 4). It was found that among the 11 subfamilies, 3 of the 33 MIKC-type PpMADS-box proteins belonged to BS, 1 belonged to AGL17, 8 belonged to AP3/P2, 3 belonged to AP1, 5 belonged to SEP, 6 belonged to SOC, and 7 belonged to AG (Fig. 4A). Among the 11 Type I-type PpMADS-box proteins, 7 belonged to Mγ and 4 belonged to Mα (Fig. 4B). Our analysis found that the 44 MADS-box proteins in Kentucky bluegrass lacked subfamilies such as FLC, SVP, AGL6, MIKC* and Mβ, which may be that the Genome-Wide of Kentucky bluegrass has not yet been obtained. Therefore, the study was based on the transcriptome to preliminary identification of the PpMADS-box genes, so its number was not complete.

Fig. 4.

Fig. 4

Open in a new tab

Phylogenetic tree analysis of MADS-box proteins from A. thaliana, O. sativa and P. pratensis. A: Phylogenetic tree of MIKC-type; B: Phylogenetic tree of Type I-type

Analysis of the expression pattern of PpMADS-box genes in diverse organs and different developmental stages

To further predict the potential of PpMADS-box genes, 12 genes were randomly selected to determine their expression patterns in different tissues at five developmental stages (Fig. 5, S3, S4 and Table S2). PpMADS20 showed no or very low expression and was not further analyzed, and PpMADS18 was only expressed in root. Among others, 9 belonged to the MIKC-type and the PpMADS27 to Type I-type. The expression trends of PpMADS-box genes with developmental stages were related to different tissues (Fig. 5, S3). PpMADS3 was downregulated during all the last four developmental stages in leaves, while the PpMADS27 was exactly the opposite. PpMADS15, PpMADS17, PpMADS36 and PpMADS38 showed similar trends with developmental stages. The difference was that PpMADS15 and PpMADS36 were upregulated only in L2, and the remaining two were upregulated in L2 and L3. PpMADS16 and PpMADS24 were upregulated only in L3. PpMADS30 was not expressed in L5. PpMADS40 was upregulated in L2, L3 and L4. The expression patterns of PpMADS-box genes in the inflorescence were relatively single, 7 genes (PpMADS3, 15, 16, 17, 36, 38, 40) had similar expression pattern, which were significantly upregulated in I5. PpMADS24 was upregulated in L2 and L3, and not expressed in the latter two stages. PpMADS27 and PpMADS30 were not expressed in I5, and only PpMADS27 was downregulated in I4. The expression pattern of PpMADS-box genes in the root was complex and changeable, and each gene had its unique expression trend. Among these, PpMADS3 was not expressed in R5, while PpMADS30 was not expressed in R1.

Fig. 5.

Fig. 5

Open in a new tab

The qRT-PCR analysis of 10 PpMADS-box genes in diverse organs at different developmental stages. The data in heatmap were calculated by ∆Ct method. I1-I5 represent respectively inflorescences samples at the booting stage, pre-anthesis, anthesis, post-anthesis and seed filling stage. L1-L5 represent respectively leaves samples at the corresponding five stages of inflorescence, and R1-R5 respectively represent roots samples at the corresponding five stages of inflorescence

The expression pattern of PpMADS-box genes in different tissues was associated with developmental stages (Fig. 5, S4). Except for PpMADS30 which was downregulated in both inflorescence and root, and PpMADS36 was downregulated in root, other 8 genes were upregulated in both inflorescence and root compared to leaf at first developmental stage. At second developmental stage, except for PpMADS36 was downregulated in root, remaining 9 genes were also upregulated in both inflorescence and root. All 10 expressed genes were upregulated in both inflorescence and root compared to the leaf at third developmental stage. Among these, 4 genes (PpMADS16, 24, 36, 38) showed higher expression in the inflorescence, and others showed higher expression in the root. At fourth developmental stage, except for PpMADS27 was not expressed in inflorescence, others were upregulated in both inflorescence and root. And PpMADS15, PpMADS24, and PpMADS40 were highly expressed in roots. PpMADS15 and PpMADS24 were not expressed in inflorescence at fifth developmental stages, and were abundantly expressed in root. Remaining 7 genes were upregulated in both inflorescence and roots compared to the leaf, and were highly and abundantly expressed in inflorescence.

Transcriptome data analysis of PpMADS-box gene family

Analysis of the expression patterns of PpMADS-box genes was based on the FPKM values in transcriptome. The materials used for transcriptome sequencing were wild germplasm Kentucky bluegrass collected in Gannan and Longnan, Gansu. The previous study proved that their apomictic rates were significantly different and can be used to study apomixes (Zhang and Ma 2022). Expression patterns of genes can provide important basis for gene functions, and the expression levels of 44 PpMADS-box genes were extracted from the RNA-seq database for inflorescence at PreM, M, PostM, and A four stages and analyzed for their expression patterns (Fig. 3B, Table S3). It was found that the 44 PpMADS-box genes could be classified into 8 categories based on their expression patterns in inflorescence. The first category contained 5 genes, PpMADS1, PpMADS4, PpMADS18, PpMADS6, and PpMADS42, which were differentially expressed in 2 genotypes. They all were upregulated at PostM in Longnan, while significantly upregulated at meiosis and PostM in Gannan. The second class contained PpMADS8, PpMADS10, PpMADS9, PpMADS11, and PpMADS31, which were mainly upregulated and expressed at the PostM in GN. The expression patterns of PpMADS44, PpMADS3, PpMADS15 and PpMADS29 in the third category and PpMADS30, PpMADS136, PpMADS25, and PpMADS26 in the fourth category were the same in the two genotypes, with the former being significantly upregulated at anthesis and the latter being significantly upregulated at PreM. The sixth category had only one gene viz PpMADS38, which was obviously upregulated in the LPreM and GA. Except for the upregulated expression of PpMADS12 and PpMADS40 in GPreM, the genes in seventh and eighth categories were only upregulated in Longnan.

Discussion

MADS-box genes are essential regulators of plant growth and development, especially during the flower development (Zhang et al. 2021b). Additionally, it was proved that the MADS-box family could be a key resource for promoting transgenic crops and traditional breeding to increase yield (Song et al. 2021; Zhao et al. 2021b). In this study, 33 MIKC-type and 11 Type I-type PpMADS-box genes were identified for the first time, and their sequences were analyzed to provide important information for studying PpMADS-box participation in inflorescence development of grass. And this paper provides basic data for improving yield through transgenic means. Furthermore, it is essential for the follow-up study of PpMADS-box and other gene families, and it is also of great significance for the improvement of the genetic characteristics of Kentucky bluegrass.

Various protein physicochemical, motif, peptide, structural analysis reveal functional diversity of the PpMADS-box proteins

To illustrate the function of PpMADS-box proteins, various protein physicochemical, motif, peptide, structural analysis were performed, and it was found that PpMADS-box proteins had diverse characteristics (Tables 1–2, Figs. 1, 2, 3 and 4a). Our results showed that the 44 PpMADS-box proteins had different physicochemical properties (Table 1), and the proportion of α-helix and random curl in the secondary structure were relatively high (Table 2), while the tertiary structure contained a helix-fold-helix structure (Fig. 4a). Properties and structure determine the gene function, so the results of this study reveal functional diversity of the PpMADS-box proteins. The analysis of signal peptide, leading peptide and transmembrane structural domain revealed that PpMADS2 and 11 each had one signal peptide, PpMADS6 and 27 each had one leading peptide, and PpMADS19 and 40 each had one transmembrane structure, respectively. The remaining proteins did not contain any signal peptides and leading peptides, nor any transmembrane structure (Table 2, Fig. 3). Subcellular localization analysis showed that except for PpMADS1 and 7) localized in mitochondria, PpMADS28 and 29  localized in the Golgi apparatus, PpMADS43 in the plasma membrane, and the others were localized in the nucleus (Table 1). Combined with all above results, it is demonstrated that the remaining 33 PpMADS-box proteins without any signal peptide, leading peptide, transmembrane structure and located in the nucleus were not transported or secreted, and directly played transcriptional regulatory roles in the nucleus. Moreover, we found that most of the MIKC-type PpMADS-box proteins contained motif1 or motif2. Except for PpMADS21 only contained motif4, the other 10 Type I-type MADS-box proteins contained motif1. Different structural domains are often associated with different functions, so our result reflected the functional specificity and diversity of PpMADS-box proteins.

The classification analysis of PpMADS-box genes demonstrates functional specificity of the PpMADS-box proteins

The number of Type I-type MADS-box are small in plants, and the current study suggested that Type I-type MADS-box genes were important regulators of plant growth and reproductive processes, such as participation in female gametophytes, embryo and endosperm, as well as seed formation and development (Li et al. 2020; Qiu and Claudia 2021). Since the transcriptome data were derived from inflorescences of two wild germplasm materials of Kentucky bluegrass at four developmental stages, and the inflorescence of grass contain spikelets with varying numbers of bracts and florets, which is the carrier of formation and development of gametophyte, embryo and seed, 11 Type I-type MADS-box genes identified in this study (Table 1) not only regulated inflorescence morphology, but also might be involved in development of megaspore, microspore, male and female gametophytes. Alhindi and Al-Abdallat (2021) reported that there was a lack of Mβ-type PpMADS-box genes in several plant species, indicating that existing MADS-box genes in these plants had a functional redundancy and other proteins can still fill their role. We also found that PpMADS (3, 4, 7, 22, 27, 42, 44) belonged to Mγ, PpMADS (1, 6, 21, 23) belonged to Mα (Fig. 5), and the lack of Mβ-type. However, the PpMADS-box genes were identified from a RNA-seq dataset collected during female gametophyte development, which might miss those MADS-box genes that are specifically expressed in the seeds, such as many of the Type I-type MADS-box genes. So, whether PpMADS-box genes had a functional redundancy is not clear.

The MIKC-type MADS-box genes can be divided into 15 subclasses, and except for a few genes, most of the A, B, C, D, and E homologous genes are MIKCC-type MADS-box genes (Song et al. 2021). According to ABCDE model, the class A genes determine lemma and palea, the class B genes specify lodicule and stamen, the class C and D genes specify stamen and ovule, and the class E genes specify organ identity and determine the spikelet meristem (Jiao et al. 2019). In this study, 33 MIKC-type PpMADS-box genes were identified, among which, PpMADS (12, 25, 26, 36, and 37) were SEP and belonged to class E (Fig. 5), indicating that they may regulate spikelet development in Kentucky bluegrass. PpMADS (8, 9, 10, 11, 29, 33 and 44) were the AG gene and belonged to the class C (Fig. 5), indicating they may regulate the stamens. PpMADS (2, 5, 13, 14, 19, 20, 30 and 42) were the AP3/PI gene, which belonged to the class B, and may determine lodicules and stamen development in Kentucky bluegrass. PpMADS (17, 18 and 38) were AP1 genes belonging to class A, which may regulate the lemma and palea, and determine the specificity of meristems in P. pratensis. Furthermore, PpMADS (16, 24, 28, 31, 39 and 40) belonged to the SOC1 genes, and previous studies reported that SOC1 acted not only to regulate the flowering time of plants (Willige et al. 2007), but also as potential marker during reproductive transition (Ahsan et al. 2019). PpMADS (32, 34 and 35) belonged to the BS genes, and PpMADS15 belonged to the AGL17 gene. These subtypes were of great significance for the establishment of flower morphology and the regulation of flowering time (Li et al. 2015; Yang et al. 2019). In general, our results preliminarily suggested that the floral organ development of Kentucky bluegrass is closely related to the expression of PpMADS-box genes.

The expression pattern of PpMADS-box genes at diverse development stages of different organs

The MADS-box genes also participate in plant root morphology and tuber development (Pérez-Ruiz et al. 2015; García-Cruz et al. 2016; Gao et al. 2018, 2020). In this study, 12 PpMADS-box genes were selected to analyze their expression patterns in diverse organs (leaf, inflorescence, and root) at different stages (Figs. 5, S3 and S4). It was found that PpMADS20 showed no or very low expression, and PpMADS18 was only expressed in root, indicating that the function of PpMADS20 in the development process of Kentucky bluegrass is not obvious, while PpMADS18 mainly has a key regulatory effect on root development. At different developmental stages, expression patterns of PpMADS-box in leaf and inflorescence were single, with complex and diverse in the roots (Fig. S3). Most selected PpMADS-box genes were highly expressed mainly in young leaves, suggesting that leaf growth was rapid during early stage and almost stopped late. In the inflorescence, most selected genes were mainly highly expressed at later stages, and it is speculated that these genes had less impact on floral organ formation and differentiation, and its function were mainly related to embryo development. Comparing the expression patterns of PpMADS-box genes in diverse organs found that except for PpMADS30 being highly expressed in the leaves, others were significantly highly expressed in inflorescence and/ or roots, indicating that the role of PpMADS-box in inflorescence and root were more important than in leaf (Fig. S4). Moreover, in the first three periods, most selected genes had higher expression in the root, suggesting that the PpMADS-box genes were mainly involved in the root development during the early stage. In the latter two stages, most selected genes had higher expression in the inflorescence, which was consistent with the abovementioned analysis in different inflorescence development stages, indicating that these genes were involved in the seed formation and development in Kentucky bluegrass.

The PpMADS-box genes may be involved in the regulation of apomixis in Kentucky bluegrass

In some particular reproductive development processes, such as somatic embryo development and parthenocarpy, MADS-box genes also play critical roles (Liu et al. 2018; Gong et al. 2021). Special reproductive modes also include apomixis reproduction, as recent studies have found that ZbAGL11 was highly expressed during the critical period of nucellar embryo development in sporophytic apomixis Zanthoxylum bungeanum, which showed that ZbAGL11 was involved in sporophytic apomixis (Fei et al. 2021). In this study, the two genotypes sequenced by the transcriptome were two wild germplasm materials of Kentucky bluegrass collected from different regions (Gannan and Longnan) of Gansu Province of China, respectively. And their apomixis rate were 11.2% and 71.4%, respectively (Zhang and Ma 2022). Analysis of the expression patterns of the PpMADS-box genes in two genotypes at four developmental stages revealed that 44 PpMADS-box genes were divided into eight classes. Except for the expression patterns of 8 genes contained in third and fourth categories were the same in both two genotypes, the remaining 36 PpMADS-box genes were specifically expressed in different genotypes at different developmental stages. Therefore, it was speculated that these genes may be vital for formation and involved in the development of apomixis in Kentucky bluegrass. However, the specific regulatory roles of PpMADS-box genes in apomixis of Kentucky bluegrass are not clear, which could be obtained by manipulating genetic manipulation utilising, for example, over-expression, gene silencing, RNAi and so on.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (JPG 2070 KB) (2MB, jpg)
Supplementary file2 (JPG 756 KB) (756KB, jpg)
Supplementary file3 (JPG 1140 KB) (1.1MB, jpg)
Supplementary file4 (JPG 1318 KB) (1.3MB, jpg)
Supplementary file5 (XLSX 16 KB) (16.1KB, xlsx)
Supplementary file6 (XLSX 9 KB) (9.4KB, xlsx)
Supplementary file7 (XLSX 12 KB) (11.8KB, xlsx)

Acknowledgements

We are grateful to the National Natural Science Foundation of China (NSFC) (project #31760699), and the "Innovation Star" Project for Outstanding Postgraduates in Gansu Province (No. 2021CXZX-347).

Authors Contributions

HM conceived the original research plans, supervised the experiments, provided funding supporting, and agreed to serve as the author responsible for contact and communication. JZ performed the experiments, analyzed the data and wrote the article.

Funding

This research was supported by the National Natural Science Foundation of China (NSFC) (project #31760699), and the "Innovation Star" Project for Outstanding Postgraduates in Gansu Province (No. 2021CXZX-347).

Declarations

Conflict of Interest

The authors declare no conflict of interest.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

  1. Ahsan MU, Hayward A, Irihimovitch V, Fletcher S, Tanurdzic M, Pocock A, Beveridge CA, Mitter N. Juvenility and vegetative phase transition in tropical/subtropical tree crops. Front Plant Sci. 2019;10:729. doi: 10.3389/fpls.2019.00729. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Alhindi T, Al-Abdallat AM. Genome-wide identification and analysis of the MADS-Box gene family in American Beautyberry (Callicarpa americana) Plants (basel, Switzerland) 2021;10(9):1805. doi: 10.3390/plants10091805. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Alvarez-Buylla ER, Pelaz S, Liljegren SJ, Gold SE, Burgeff C, Ditta GS, Ribas de Pouplana L, Martínez-Castilla L, Yanofsky MF. An ancestral MADS-box gene duplication occurred before the divergence of plants and animals. Proc Natl Acad Sci USA. 2000;97(10):5328–5333. doi: 10.1073/pnas.97.10.5328. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Arora R, Agarwal P, Ray S, Singh AK, Singh VP, Tyagi AK, Kapoor S. MADS-box gene family in rice: genome-wide identification, organization and expression profiling during reproductive development and stress. BMC Genomics. 2007;8:242. doi: 10.1186/1471-2164-8-242. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bowman JL, Smyth DR, Meyerowitz EM. Genetic interactions among floral homeotic genes of Arabidopsis. Development (cambridge, England) 1991;112(1):1–20. doi: 10.1242/dev.112.1.1. [DOI] [PubMed] [Google Scholar]
  6. Chandler JW. Founder cell specification. Trends Plant Sci. 2011;16(11):607–613. doi: 10.1016/j.tplants.2011.08.005. [DOI] [PubMed] [Google Scholar]
  7. Chen C, Chen H, Zhang Y, Thomas HR, Frank MH, He Y, Xia R. TBtools: an integrative toolkit developed for interactive analyses of big biological data. Mol Plant. 2020;13(8):1194–1202. doi: 10.1016/j.molp.2020.06.009. [DOI] [PubMed] [Google Scholar]
  8. Chen F, Zhang X, Liu X, Zhang L. Evolutionary analysis of MIKC(c)-Type MADS-Box genes in gymnosperms and angiosperms. Front Plant Sci. 2017;8:895. doi: 10.3389/fpls.2017.00895. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chen M, Nie G, Yang L, Zhang Y, Cai Y. Homeotic transformation from stamen to petal in Lilium is associated with MADS-box genes and hormone signal transduction. Plant Growth Regul. 2021;95(1):49–64. doi: 10.1007/s10725-021-00724-6. [DOI] [Google Scholar]
  10. Cho LH, Yoon J, An G. The control of flowering time by environmental factors. Plant J Cell Mol Biol. 2017;90(4):708–719. doi: 10.1111/tpj.13461. [DOI] [PubMed] [Google Scholar]
  11. Chopy M, Cavallini-Speisser Q, Chambrier P, Morel P, Just J, Hugouvieux V, Bento S, Zubieta C, Vandenbussche M, Monniaux M (2021) Cell layer-specific expression of the B-class MADS-box gene PhDEF drives petal tube or limb development in petunia flowers. doi:10.1101/2021.04.03.438311 [DOI] [PMC free article] [PubMed]
  12. Cullerne DP, Fjellheim S, Spriggs A, Eamens AL, Trevaskis B, Wood CC. A vernalization response in a winter safflower (Carthamus tinctorius) involves the upregulation of homologs of FT, FUL, and MAF. Front Plant Sci. 2021;12:639014. doi: 10.3389/fpls.2021.639014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Dixon LE, Greenwood JR, Bencivenga S, Zhang P, Cockram J, Mellers G, Ramm K, Cavanagh C, Swain SM, Boden SA. TEOSINTE BRANCHED1 regulates inflorescence architecture and development in bread wheat (Triticum aestivum) Plant Cell. 2018;30(3):563–581. doi: 10.1105/tpc.17.00961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fei X, Shi Q, Qi Y, Wang S, Lei Y, Hu H, Liu Y, Yang T, Wei A. ZbAGL11, a class D MADS-box transcription factor of Zanthoxylum bungeanum, is involved in sporophytic apomixis. Hortic Res. 2021;8(1):23. doi: 10.1038/s41438-020-00459-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gao H, Wang Z, Li S, Hou M, Zhou Y, Zhao Y, Li G, Zhao H, Ma H. Genome-wide survey of potato MADS-box genes reveals that StMADS1 and StMADS13 are putative downstream targets of tuberigen StSP6A. BMC Genomics. 2018;19(1):726. doi: 10.1186/s12864-018-5113-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gao YR, Sun JC, Sun ZL, Xing Y, Zhang Q, Fang KF, Cao QQ, Qin L. The MADS-box transcription factor CmAGL11 modulates somatic embryogenesis in Chinese chestnut (Castanea mollissima Blume) J Integr Agric. 2020;19(4):1033–1043. doi: 10.1016/S2095-3119(20)63157-4. [DOI] [Google Scholar]
  17. García-Cruz KV, García-Ponce B, Garay-Arroyo A, Sanchez MP, Ugartechea-Chirino Y, Desvoyes B, Pacheco-Escobedo MA, Tapia-López R, Ransom-Rodríguez I, Gutierrez C, Alvarez-Buylla ER. The MADS-box XAANTAL1 increases proliferation at the Arabidopsis root stem-cell niche and participates in transition to differentiation by regulating cell-cycle components. Ann Bot. 2016;118(4):787–796. doi: 10.1093/aob/mcw126. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gong P, Song C, Liu H, Li P, Zhang M, Zhang J, Zhang S, He C. Physalis floridana CRABS CLAW mediates neofunctionalization of GLOBOSA genes in carpel development. J Exp Bot. 2021;72(20):6882–6903. doi: 10.1093/jxb/erab309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Gramzow L, Theißen G. Phylogenomics reveals surprising sets of essential and dispensable clades of MIKC(c)-group MADS-box genes in flowering plants. J Exp Zool B Mol Dev Evol. 2015;324(4):353–362. doi: 10.1002/jez.b.22598. [DOI] [PubMed] [Google Scholar]
  20. Hussin SH, Wang H, Tang S, Zhi H, Tang C, Zhang W, Jia G, Diao X. SiMADS34, an E-class MADS-box transcription factor, regulates inflorescence architecture and grain yield in Setaria italica. Plant Mol Biol. 2021;105(4–5):419–434. doi: 10.1007/s11103-020-01097-6. [DOI] [PubMed] [Google Scholar]
  21. Jiao Z, Li J, Ni Y, Jiang Y, Sun Y, An J, Li H, Zhang J, Hu X, Li Q, Niu J. Enhanced senescence process is the major factor stopping spike differentiation of wheat mutant ptsd1. Int J Mol Sci. 2019;20(18):4642. doi: 10.3390/ijms20184642. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kater MM, Dreni L, Colombo L. Functional conservation of MADS-box factors controlling floral organ identity in rice and Arabidopsis. J Exp Bot. 2006;57(13):3433–3444. doi: 10.1093/jxb/erl097. [DOI] [PubMed] [Google Scholar]
  23. Kong L, Duan Y, Ye Y, Cai Z, Wang F, Qu X, Qiu R, Wu C, Wu W. Screening and analysis of proteins interacting with OsMADS16 in rice (Oryza sativa L.) PLoS ONE. 2019;14(8):e0221473. doi: 10.1371/journal.pone.0221473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kumar K, Srivastava H, Das A, Tribhuvan KU, Durgesh K, Joshi R, Sevanthi AM, Jain PK, Singh NK, Gaikwad K. Identification and characterization of MADS box gene family in pigeonpea for their role during floral transition. 3 Biotech. 2021;11(2):108. doi: 10.1007/s13205-020-02605-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kwantes M, Liebsch D, Verelst W. How MIKC* MADS-box genes originated and evidence for their conserved function throughout the evolution of vascular plant gametophytes. Mol Biol Evol. 2012;29(1):293–302. doi: 10.1093/molbev/msr200. [DOI] [PubMed] [Google Scholar]
  26. Li F, Fan G, Lu C, Xiao G, Zou C, Kohel RJ, Ma Z, Shang H, Ma X, Wu J, Liang X, Huang G, Percy RG, Liu K, Yang W, Chen W, Du X, Shi C, Yuan Y, Ye W, Liu X, Zhang X, Liu W, Wei H, Wei S, Huang G, Zhang X, Zhu S, Zhang H, Sun F, Wang X, Liang J, Wang J, He Q, Huang L, Wang J, Cui J, Song G, Wang K, Xu X, Yu JZ, Zhu Y, Yu S. Genome sequence of cultivated Upland cotton (Gossypium hirsutum TM-1) provides insights into genome evolution. Nat Biotechnol. 2015;33(5):524–530. doi: 10.1038/nbt.3208. [DOI] [PubMed] [Google Scholar]
  27. Li SY, Zhang Q, Jin YH, Zou JX, Zheng YS, Li DD. A MADS-box gene, EgMADS21, negatively regulates EgDGAT2 expression and decreases polyunsaturated fatty acid accumulation in oil palm (Elaeis guineensis Jacq.) Plant Cell Rep. 2020;39(11):1505–1516. doi: 10.1007/s00299-020-02579-z. [DOI] [PubMed] [Google Scholar]
  28. Liu L, Wang Z, Liu J, Liu F, Zhai R, Zhu C, Wang H, Ma F, Xu L. Histological, hormonal and transcriptomic reveal the changes upon gibberellin-induced parthenocarpy in pear fruit. Horticul Res. 2018;5:1. doi: 10.1038/s41438-017-0012-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Ma JJ, Chen X, Song YT, Zhang GF, Zhou XQ, Que SP, Mao F, Pervaiz T, Lin JX, Li Y, Li W, Wu HX, Niu SH. MADS-box transcription factors MADS11 and DAL1 interact to mediate the vegetative-to-reproductive transition in pine. Plant Physiol. 2021;187(1):247–262. doi: 10.1093/plphys/kiab250. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Moser M, Asquini E, Miolli GV, Weigl K, Hanke MV, Flachowsky H, Si-Ammour A. The MADS-Box gene MdDAM1 controls growth cessation and bud dormancy in apple. Front Plant Sci. 2020;11:1003. doi: 10.3389/fpls.2020.01003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Parenicová L, de Folter S, Kieffer M, Horner DS, Favalli C, Busscher J, Cook HE, Ingram RM, Kater MM, Davies B, Angenent GC, Colombo L. Molecular and phylogenetic analyses of the complete MADS-box transcription factor family in Arabidopsis: new openings to the MADS world. Plant Cell. 2003;15(7):1538–1551. doi: 10.1105/tpc.011544. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Pérez-Ruiz RV, García-Ponce B, Marsch-Martínez N, Ugartechea-Chirino Y, Villajuana-Bonequi M, de Folter S, Azpeitia E, Dávila-Velderrain J, Cruz-Sánchez D, Garay-Arroyo A, Sánchez Mde L, Estévez-Palmas JM, Álvarez-Buylla ER. XAANTAL2 (AGL14) Is an important component of the complex gene regulatory network that underlies arabidopsis shoot apical meristem transitions. Mol Plant. 2015;8(5):796–813. doi: 10.1016/j.molp.2015.01.017. [DOI] [PubMed] [Google Scholar]
  33. Qiu Y, Claudia K. Endosperm evolution by duplicated and neofunctionalized Type I MADS-box transcription factors. Mol Biol Evol. 2021 doi: 10.1101/2021.06.08.447573. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Qu Y, Bi C, He B, Ye N, Yin T, Xu LA. Genome-wide identification and characterization of the MADS-box gene family in Salix suchowensis. PeerJ. 2019;7:e8019. doi: 10.7717/peerj.8019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Schilling S, Kennedy A, Pan S, Jermiin LS, Melzer R. Genome-wide analysis of MIKC-type MADS-box genes in wheat: pervasive duplications, functional conservation and putative neofunctionalization. New Phytol. 2020;225(1):511–529. doi: 10.1111/nph.16122. [DOI] [PubMed] [Google Scholar]
  36. Sharma N, Geuten K, Giri BS, Varma A. The molecular mechanism of vernalization in Arabidopsis and cereals: role of Flowering Locus C and its homologs. Physiol Plant. 2020;170(3):373–383. doi: 10.1111/ppl.13163. [DOI] [PubMed] [Google Scholar]
  37. Singh S, Rana V, Kumar A, Kumar A, Kumar P. MOLECULAR CLONING AND CHARACTERIZATION OF FBP21 (FLORAL BINDING PROTEIN 21) MADS-BOX GENE IN PETUNIA (Petunia axillaris) Plant Cell Biotechnol Mol Biol. 2021;22:30–45. [Google Scholar]
  38. Song GQ, Han X, Ryner JT, Thompson A, Wang K. Utilizing MIKC-type MADS-box protein SOC1 for yield potential enhancement in maize. Plant Cell Rep. 2021;40(9):1679–1693. doi: 10.1007/s00299-021-02722-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Sri T, Gupta B, Tyagi S, Singh A. Homeologs of Brassica SOC1, a central regulator of flowering time, are differentially regulated due to partitioning of evolutionarily conserved transcription factor binding sites in promoters. Mol Phylogenet Evol. 2020;147:106777. doi: 10.1016/j.ympev.2020.106777. [DOI] [PubMed] [Google Scholar]
  40. Sun CH, Wang JH, Gu KD, Zhang P, Zhang XY, Zheng CS, Hu DG, Ma F. New insights into the role of MADS-box transcription factor gene CmANR1 on root and shoot development in chrysanthemum (Chrysanthemum morifolium) BMC Plant Biol. 2021;21(1):79. doi: 10.1186/s12870-021-02860-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Theissen G. Development of floral organ identity: stories from the MADS house. Curr Opin Plant Biol. 2001;4(1):75–85. doi: 10.1016/s1369-5266(00)00139-4. [DOI] [PubMed] [Google Scholar]
  42. Theißen G, Gramzow L. Chapter 8 - structure and evolution of plant MADS domain transcription factors. In: Gonzalez DH, editor. Plant transcription factors. Boston: Academic Press; 2016. pp. 127–138. [Google Scholar]
  43. Theißen G, Melzer R, Rümpler F. MADS-domain transcription factors and the floral quartet model of flower development: linking plant development and evolution. Development (cambridge, England) 2016;143(18):3259–3271. doi: 10.1242/dev.134080. [DOI] [PubMed] [Google Scholar]
  44. Trapnell C, Williams BA, Pertea G, Mortazavi A, Kwan G, van Baren MJ, Salzberg SL, Wold BJ, Pachter L. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotechnol. 2010;28(5):511–515. doi: 10.1038/nbt.1621. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Wen X, Qi S, Huang H, Wu X, Zhang B, Fan G, Yang L, Hong Y, Dai S. The expression and interactions of ABCE-class and CYC2-like genes in the capitulum development of Chrysanthemum lavandulifolium and C. × morifolium. Plant Growth Regul. 2019 doi: 10.1007/s10725-019-00491-5. [DOI] [Google Scholar]
  46. Willige BC, Ghosh S, Nill C, Zourelidou M, Dohmann EM, Maier A, Schwechheimer C. The DELLA domain of GA INSENSITIVE mediates the interaction with the GA INSENSITIVE DWARF1A gibberellin receptor of Arabidopsis. Plant Cell. 2007;19(4):1209–1220. doi: 10.1105/tpc.107.051441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Wu D, Liang W, Zhu W, Chen M, Ferrándiz C, Burton RA, Dreni L, Zhang D. Loss of LOFSEP transcription factor function converts spikelet to leaf-like structures in rice. Plant Physiol. 2018;176(2):1646–1664. doi: 10.1104/pp.17.00704. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Yang X. Analysis of the copy number of exogenous genes in transgenic cotton using real-time quantitative PCR and the 2-△△CT method. African J Biotechnol 11. 2012 doi: 10.5897/AJB11.4117. [DOI] [Google Scholar]
  49. Yang Z, Ge X, Yang Z, Qin W, Sun G, Wang Z, Li Z, Liu J, Wu J, Wang Y, Lu L, Wang P, Mo H, Zhang X, Li F. Extensive intraspecific gene order and gene structural variations in upland cotton cultivars. Nat Commun. 2019;10(1):2989. doi: 10.1038/s41467-019-10820-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Zhang JQ, Ma HL. The female gametophyte characteristics and gene expression analysis involved in Apomixis of wild germplasm materials of kentucky bluegrass in gansu province of china. J Plant Growth Regul. 2022 doi: 10.1007/s00344-022-10702-5. [DOI] [Google Scholar]
  51. Zhang JQ, Niu KJ, Li YZ, MA HL, Factors inducing plant apomixis and its potential application in Kentucky bluegrass breeding. Acta Pratacul Sin. 2021;30(4):205–217. doi: 10.11686/cyxb2020176. [DOI] [Google Scholar]
  52. Zhang L, Zhang H, Qiao L, Miao L, Yan D, Liu P, Zhao G, Jia J, Gao L. Wheat MADS-box gene TaSEP3-D1 negatively regulates heading date. Crop J. 2021 doi: 10.1016/j.cj.2020.12.007. [DOI] [Google Scholar]
  53. Zhang ZB, Jin YJ, Wan HH, Cheng L, Feng ZG. Genome-wide identification and expression analysis of the MADS-box transcription factor family in Camellia sinensis. J Appl Genet. 2021;62(2):249–264. doi: 10.1007/s13353-021-00621-8. [DOI] [PubMed] [Google Scholar]
  54. Zhao J, Gong P, Liu H, Zhang M, He C. Multiple and integrated functions of floral C-class MADS-box genes in flower and fruit development of Physalis floridana. Plant Mol Biol. 2021;107(1–2):101–116. doi: 10.1007/s11103-021-01182-4. [DOI] [PubMed] [Google Scholar]
  55. Zhao W, Zhang LL, Xu ZS, Fu L, Pang HX, Ma YZ, Min DH. Genome-Wide Analysis of MADS-Box Genes in Foxtail Millet (Setaria italica L.) and Functional Assessment of the Role of SiMADS51 in the Drought Stress Response. Front Plant Sci. 2021;12:659474. doi: 10.3389/fpls.2021b.659474. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Zhou F-Y, Han Y-J, Wang Y-H, Yao C-C, Zhang Y. Overexpression of Polypogon fugax Type I-Like MADS-Box gene PfAGL28 affects flowering time and pod formation in transgenic arabidopsis. Plant Mol Biol Report. 2021 doi: 10.1007/s11105-021-01312-8. [DOI] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary file1 (JPG 2070 KB) (2MB, jpg)
Supplementary file2 (JPG 756 KB) (756KB, jpg)
Supplementary file3 (JPG 1140 KB) (1.1MB, jpg)
Supplementary file4 (JPG 1318 KB) (1.3MB, jpg)
Supplementary file5 (XLSX 16 KB) (16.1KB, xlsx)
Supplementary file6 (XLSX 9 KB) (9.4KB, xlsx)
Supplementary file7 (XLSX 12 KB) (11.8KB, xlsx)

Articles from Physiology and Molecular Biology of Plants are provided here courtesy of Springer

RESOURCES