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Tracing the footprints of the ABCDE model of flowering in Phalaenopsis equestris (Schauer) Rchb.f. (Orchidaceae)
J Plant Biotechnol 2019;46:255-273
Published online December 31, 2019
© 2019 Korean Society for Plant Biotechnology.

Himani · Thakku R. Ramkumar · Shivi Tyagi · Himanshu Sharma · Santosh K. Upadhyay · Jaspreet K. Sembi

Department of Botany, Panjab University, Chandigarh, India
Agronomy department, IFAS, University of Florida, Gainesville, FL, 32611, USA
IKG Punjab Technical University, Jalandhar
Correspondence to: e-mail: jaspreet.sembi@pu.ac.in
Received August 13, 2019; Revised December 23, 2019; Accepted December 23, 2019.
cc This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract

Orchids are indispensable to the floriculture industry due to their unique floral organization. The flowers have two outer whorls of tepals including a lip (labellum), and two inner whorls, pollinia and gynostemiun (column). The floral organization and development is controlled at the molecular level, mainly by the MADS-box gene family, comprising homeotic genes divided into type I and type II groups. The type I group has four sub-groups, Mα, Mβ, Mγ, and Mδ, playing roles in seed, embryo, and female reproductive organ development; the type II group genes form classes A, B, C, D, and E, which are a part of the MIKCC subgroup with specific roles in florigenesis and organization. The coordinated functioning of these classes regulates the development of various floral whorls. The availability of genome and transcriptome sequence data for Phalaenopsis equestris offers an opportunity to validate the ABCDE model of flower development. Hence, this study sought to characterize the MADS-box gene family and elucidate of the ABCDE model. A total of 48 identified MADS-box proteins, including 20 type I [Mα (12), Mγ (8)] and 28 type II [MIKCC (27), MIKC* (1)] members, were characterized for physico-chemical features and domains and motifs organization. The exon-intron distribution and the upstream cis-regulatory elements in the promoter regions of MADS-box genes were also analysed. The discrete pace of duplication events in type I and type II genes suggested differential evolutionary constraints between groups. The correlation of spatio-temporal expression pattern with the presence of specific cis-regulatory elements and putative protein–protein interaction within the different classes of MADS-box gene family endorse the ABCDE model of floral development.

Keywords : MADS-box genes, Phalaenopsis equestris, Characterization, Expression, Flowering, ABCDE model
Introduction

The MADS-box genes family encode transcription factors that partake in numerous developmental processes and signal transductions (Riechmann and Meyerowitz 1997; Shore and Sharrocks 1995; Theissen et al. 1996, 2000). The name MADS-box is derived from four different proteins from taxonomically diverge species, namely MINI CHROMOSOME MAINTENANCE 1 (M) of Sacchromyces cerevisiae (Passmore et al. 1988), AGAMOUS (A) of Arabidopsis thaliana (Yanofsky et al. 1990), DEFICIENS (D) of Antirrhinum majus (Sommer et al. 1990) and SERUM RESPONSIVE FACTOR (S) of Homo sapiens (Norman et al. 1988). The pivotal role of MADS-box genes in florigenesis was initially established upon studying the genetic mutants in A. thaliana and A. majus (Coen and Meyerowitz 1991). Originally, the members of MADS-box gene family were functionally characterised into three classes (A, B and C) and this laid the foundation for development of the ABC model of floral development (Coen and Meyerowitz 1991). The whorl 1 (calyx with sepals) is controlled by class A genes (APETALA1; AP1), while the whorl 2 (corolla with petals) development is controlled by a combinatorial effect of class A (APETALA3; AP3) and class B (PISTILLATA; PI) genes. The class B and class C (AGAMOUS; AG) genes regulate the development of the whorl 3 (androecium with stamens), and the interior most whorl (gynoecium with carpels) development is controlled by C class genes (Bowman et al. 1991; Coen and Meyerowitz 1991; Meyerowitz et al. 1991). The ABC model was later extended by including class D and E genes. The D-class genes (SEEDSTICK, STK and SHATTERPROOF, SHP) are shown to be involved in ovule formation in Petunia (Angenent and Colombo 1996) and the E-class genes (SEPALLATA: SEP1/2/3/4) identified in A. thaliana with roles in coordinated functioning of all the other classes genes, leading to the synchronised development of floral whorls (Pelaz et al. 2000). Thus, the ‘ABC model’ was revised to ‘ABCDE model’ (Theissen 2001).

The genomic organisations, evolutionary lineages, developmental functions and their functional redundancy led to the division of MADS-box genes into two major groups, as type I and type II (Alvarez-Buylla et al. 2000a). Parenicova et al. (2003) classified the type I MADS-box genes further into four sub-groups, Mα, Mβ, Mγ and Mδ, based on their phylogeny and conserved nature of MADS-box domain. The type I genes are involved predominantly in female gametophyte, embryo and seed development (Masiero et al. 2011). Proteins of type II MADS-box genes carry characteristic domains, MADS-box (M), intervening (I), keratin (K) and C-terminus (C), and hence often termed as MIKC-type genes. The M domain is highly conserved followed by K domain (Davies et al. 1996; Fischer et al. 1995; Ma et al. 1991; Pnueli et al. 1991) whereas, the I and C domains are least conserved (Parenicova et al. 2003). The K domain is involved in protein-protein interaction, while I domain is responsible for DNA binding and dimerization of proteins (Riechmann et al. 1996). The variable C domain is considered to play a role in transcription initiation (Davies et al. 1996; Kramer et al. 1998; Riechmann et al. 1996). The highly conserved nature of MADS-box and K domain indicates that both domains were subjected to very strong constraints, structurally as well as functionally in comparison to the less conserved I and C domains (De Bodt et al. 2003). The type II (MIKC) genes are further divided into two sub-groups MIKCC and MIKC* based on their protein structural organization (Henschel et al. 2002). MIKC* proteins consist of longer I domain and less conserved K domain than MIKCC proteins (Adamczyk and Fernandez 2009; Henschel et al. 2002). The MIKCC have been further divided phylogenetically into multiple sub-classes, 12 in A. thaliana (Parenicova et al. 2003), 13 in grape (Diaz-Riquelme et al. 2009) and 14 in Oryza sativa (Arora et al. 2007). These MIKCC proteins are reported to be involved in multiple developmental processes, especially in floral organ and fruit development (Alvarez-Buylla et al. 2000b; Kaufmann et al. 2005), whereas the MIKC* genes essentially participate in male gametophyte development (Zobell et al. 2010). Considering the genomic organization, the type I genes are either intron-less or mono-intronic, while the type II genes have multiple introns (Parenicova et al. 2003).

The characteristic N-terminus MADS-box domain (~60 aa) binds to the DNA element CArG box [CC(A/T)6GG] and forms either homodimer or heterodimer (Riechmann and Meyerowitz 1997). The dimerization is necessary for the formation of floral quartet complexes (AP1/AP1/SEP/ SEP, AP1/SEP/AP3/PI, AG/SEP/AP3/PI and AG/AG/SEP/ SEP) that induces floral organ formation (Theissen and Saedler 2001). The MIKC proteins form dimer preferentially with other type II members, and scarcely with type I members (de Folter et al. 2005). Similarly, the type I MADS proteins preferentially associate with other type I members. Within the type I proteins, Mα, Mβ, and Mγ types form heterodimer with each other. The Mα interacts predominantly with Mβ and Mγ and rarely with itself. This indicates that type I protein complexes might get stabilized by Mα and establish as higher order complexes (Immink et al. 2009). Masiero et al. (2011) have proposed that type I MADS-box factors probably do not bind to DNA.

MADS-box gene family has been characterised in several plant species across the plant kingdom from algae to angiosperms. Recent whole genome sequencing and global transcriptome profiling of floriculturally important orchids have enabled genome-wide characterization and expression profiling of important gene families. Genome-wide identification of MADS-box gene has been reported in orchids like Apostasia shenzhenica (Zhang et al. 2017), Dendrobium catenatum (Zhang et al. 2016), Dendrobium officinale (Yan et al. 2015) and P. equestris (Cai et al. 2015). Lin et al. (2016) carried out transcriptome-wide analysis and characterization of MADS-box gene family in Erycina pusilla. Functional characterization of several MADS-box genes in P. equestris have been reported earlier (Tsai et al. 2004, 2005; Chen et al. 2012). However, analyses for protein characterization, gene structure, spatio-temporal expression and upstream cis-regulatory elements of PeMADS genes at a genome-wide scale were not performed earlier. The present study is an attempt to fill this lacuna and consolidate the fragmented information available on PeMADS gene family, and further attempts to physico-chemically characterize the PeMADS proteins and elucidate the ABCDE model of flowering through development of interaction network and gene expression profiling in P. equestris.

Material and Methods

Structural analysis

The PeMADS protein sequences and CDS sequences for PeMADS genes as reported by Cai et al. (2015), were retrieved from Orchidbase 3.0 database (http://orchidbase.itps.ncku.edu.tw/). The genomic DNA and 1500 upstream promoter sequences for PeMADS genes were extracted from NCBI database (https://www.ncbi.nlm.nih.gov/). The intron-exon arrangement in PeMADS genes was drawn using GSDS - Gene Structure Display Server 2.0 (gsds.cbi.pku.edu.cn/). The PeMADS promoter sequences were analysed for occurrence of upstream cis-regulatory elements using PLACE (https://sogo.dna.affrc.go.jp/cgi-bin/sogo.cgi?lang=en&pj=640&action=page&page=newplace). The presence of MADS-box domain (PF00319) and K-box domain (PF01486) in PeMADS protein sequences was confirmed by SMART server (http://smart.embl-heidelberg.de/). The domain architecture was generated using IBS (Illustrator for biological sequences) tool (http://ibs.biocuckoo.org/online.php). The MULTALIN tool (multalin.toulouse.inra.fr/multalin/) was employed to locate the domain position by multiple sequence alignment. The conserved motifs were identified by MEME suite server (http://meme-suite.org/tools/meme) with preset parameters (maximum number of motifs - 10, number of repetitions - any, optimum motif width - ≥6 to ≤200).

Phylogenetic analysis

Full length PeMADS protein sequences were pre-aligned using inbuilt MUSCLE program and the phylogenetic tree was created using MEGA7 tool (http://www.megasoftware.net/) by neighbour-joining method at 1000 bootstrap replicates.

Protein characterization

The physico-chemical properties of PeMADS proteins like molecular weight, aliphatic index, instability index, pI and GRAVY value were calculated using PROTPARAM (http://web.expasy.org/protparam/). The sub-cellular localization of proteins was examined by CELLO v. 2.5 (http://cello.life.nctu.edu.tw/) and WoLF PSORT (http://www.genscript.com/wolf-psort.html). The presence of signal peptide and transmembrane region was explored respectively by SignalP.4.0 server (http://www.cbs.dtu.dk/services/signalp/), and TMHMM server v.2.0 (http://www.cbs.dtu.dk/services/TMHMM/). GLOBPLOT (http://globplot.embl.de/) was employed to evaluate the globularity of proteins.

Interaction network

Interaction input data was generated by analysing the structural basis of protein-protein interaction using Struct2Net tool (http://cb.csail.mit.edu/cb/struct2net/webserver/) with specific parameters (query method: Thread sequences onto all templates and struct2net prediction algorithm). The generated data was used to construct the interaction network among specific PeMADS proteins using Cytoscape tool (http://www.cytoscape.org/).

Duplication events and ortholog prediction

The sequence similarity within PeMADS CDS sequences were analysed using MAFFT tool (https://www.ebi.ac.uk/Tools/msa/mafft/) and genes sharing sequence similarity ≥80% in CDS were considered duplicated (Sharma et al. 2019). The orthologous genes for PeMADS genes were predicted by BLASTn search against MADS genes of A. thaliana (AtMADS) and O. sativa (OsMADS) retrieved from their respective databases (https://www.arabidopsis.org/; http://rice.plantbiology.msu.edu/).

Expression Analysis

Spatio-temporal expression profiling of PeMADS genes was done using RPKM values retrieved from Orchidbase 3.0 database (http://orchidbase.itps.ncku.edu.tw) for different vegetative and reproductive plant developmental stages such as leaf, root, floral stalk, flower, sepal, petal, labellum and gynostemium. The genes having null RPKM expression value in all the tissues were not included for evaluation. The heat map was generated using Hierarchical Clustering Explorer 3.5 (http://www.cs.umd.edu/hcil/hce/).

Results

Structural analysis

The P. equestris genome carries 51 MADS-box (PeMADS) genes, of which 48 genes were grouped into type I (20) [Mα (8) and Mγ (12)] and type II (28) [MIKCC (27) and MIKC* (1)]. No Mβ and Mδ type genes were reported (Cai et al. 2015). Data was insufficient for one C class gene (PeMADS1) and two Mα genes (PeMADS5 and PeMADS6), hence these were not included for further analysis. Majority (16/20) of type I genes were intron-less, while others carried small-stretch and lesser number of introns (Fig. 1a). On the other hand, type II genes harboured multiple and long-stretch introns. The single MIKC* (PeMADS9) gene had the maximum of 10 introns (Fig. 1b). The intronic phases were majorly biphasic in type I, while in type II, 0 phase introns dominated (Fig. 1).

Fig. 1.

Intron-exon arrangement in MADS-box genes. (A) Type I. (B) Type II. Exons and introns are represented as boxes and thin lines, respectively. 0 = phase 0 intron; 1 = phase 1 intron; 2 = phase 2 intron


Fig. 2.

Distribution of ten most frequently occurs cis-regulatory elements on MADS-box genes. (A) Type I. (B) Type II. Five were annotated while five remained unknown


All the PeMADS proteins carried the signature MADS-box domain (~60aa). The type II PeMADS (except PeMADS7/14/16/20/22/25) additionally carried a less conserved K-box domain (~90aa), C-terminal domain and intervening I domain (Fig. 3, 4; Table 1). Motif analysis by MEME-suite server identified 10 conserved motifs. Motif 1 representing the MADS-box was present in all the proteins. Surprisingly, one protein (PeMADS22) lacked any of the identified motifs. Motifs 3, 4, 5, 8, 9 and 10 were restricted to type I, while motif 2 and 7 were restricted to type II proteins. Motif 2, representing the K-box domain, was only present in MIKCC group of type II proteins. Motif 3 and 4 represented internal repeats, and motif 5 and 9 represented the low complexity region, while the motif 6, 7, 8 and 10 were of unknown domains (Fig. 5).

Fig. 3.

Domain Organization in MADS-box proteins. (A) Single MADS domain shown in red within type I MADS-box proteins. (B) Additional K domain in blue within type II MADS-box proteins. Green color represents the protein length


Fig. 4.

Multiple sequence alignments of MADS-box proteins (A) Type I. (B) Type II. The green box represents the MADS domain and the black over lined region represents the signal motif (KR(K/R)X4KK) required for nuclear localization. In type II, the less conserved I domain is present in between MADS and K domain and the variable C-domain at the c-terminus region. Asterisks represent the conserved amino acid residues


Fig. 5.

Organization of Conserved motifs of MADS-box proteins. (A) Type I. (B) Type II. (C) Ten conserved motif sequences of MADS-box proteins showing e-value and site count


Domain Organization in MADS-box proteins

GENE IDsMADS box DomainPFAM:kboxTransmembrane domainInternal repeat 1 (RPT)Coiled coil regionLow complexity regionUnknown region
PeMADS601-6061-175
PeMADS581-60119-130191-127861-118
175-186131-174
PeMADS571-6089-11961-88
PeMADS569-68149-191308-32369-148
192-246
295-307
247-294324-428
PeMADS551-60141-189 249-25761-140
189-239240-248
258-305
PeMADS541-6061-214
PeMADS534-63100-11164-99
112-125
PeMADS504-6183-9462-82
95-148
PeMADS484-6183-9462-82
95-148
PeMADS474-63100-11164-99
112-125
PeMADS434-6970-149
PeMADS4010-6970-116
PeMADS399-6894-176188-20369-93
117-187
204-226
PeMADS374-63147-16964-146
170-215
PeMADS341-4950-157
PeMADS331-6061-127
PeMADS321-6086-11561-85
116-151
PeMADS311-6061-111
PeMADS301-60221-22661-120
255-357227-254
358-384
PeMADS292-6162-186
PeMADS81-6080-17261-79
173-250
PeMADS71-6061-69
PeMADS61-6072-16561-71
166-210
PeMADS51-6077-16961-76
170-219
PeMADS41-6077-16961-76
170-224
PeMADS383-6281-17263-80
PeMADS361-6078-155157-17261-77
PeMADS31-6075-169174-18561-74
186-222
PeMADS281-6075-14061-74
PeMADS271-6083-18161-82
182-194
PeMADS261-6063-171172-192
PeMADS258-6770-9293-103
PeMADS246-6585-17666-84
177-219
PeMADS231-6085-17161-84
172-228
PeMADS225-5354-74
PeMADS211-6081-173230-25361-80
276-289174-229
254-275
290-301
PeMADS201-60164-18261-163
183-290
PeMADS227-86104-1956-2187-103
196-253
PeMADS191-6080-17261-79
173-222
PeMADS181-6080-17161-79
172-223
PeMADS166-6566-80
PeMADS151-6078-167
PeMADS141-60
PeMADS131-6062-18261-81
163-175
PeMADS121-6077-17261-76
173-246
PeMADS111-6079-17261-78
173-244
PeMADS101-6080-17261-79
173-244
PeMADS91-60264-289107-14261-106
143-263
290-321

Phylogenetic analysis

Phylogenetically, the type I proteins clearly diverged from the type II proteins and formed separate clades of Mα (PeMADS37/39/40/43/47/48/50/53) and Mγ (PeMADS29/ 30/31/32/33/34/54/55/56/57/58/60 (Fig. 6). On the other hand, type II proteins congregated to form clear groups pertaining to classes, A (PeMADS20/21/22), B (PeMADS2/ 3/4/5/6), C (PeMADS24/25), D (PeMADS7/38) and E (PeMADS8/10/11/12/13). The other type II proteins grouped into subclades: OsMADS32-like (PeMADS36), SVP (PeMADS23), ANR1 (PeMADS26/27), Bs (PeMADS28), SOC1 (PeMADS18/19) AGL6 (PeMADS14/16) and MIKC* (PeMADS9). Noteworthy, PeMADS15 protein did not group with other AGL6 subclade and remained an outlier.

Fig. 6.

Phylogenetic relationship among type I and type II MADS-box proteins. The MADS-box family proteins involved in floral whorls formation are grouped together


Protein characterization

PeMADS had an average length of 195aa and molecular weight of 21.92kDa that ranged from 61aa to 428aa and 7kDa to 35kDa. PeMADS56 (type I Mα) was the largest protein (428aa with 49.2kDa), while the PeMADS14 (type II MIKCC) represented the smallest protein (61 aa with 7kDa). The isoelectric point ranged from 5.9 to 9.7 in type I, and 4.9 to 10.1 in type II proteins, indicating that most of the PeMADS proteins are alkaline in nature as reported earlier in O. sativa (Arora et al. 2007). The aliphatic index value ranged from 67.7 to 101.8 in type I, and ranged from 56.5 to 106.02 in type II (File S1). All the proteins were predicted unstable except for the three proteins (PeMADS03/23/27) of MIKCC type II. The grand average of hydropathy (GRAVY) value ranged from -0.922 to 0.263 with all the proteins having a negative GRAVY value, except PeMADS37 (type I) and PeMADS22 and PeMADS25 (type II) indicated that most of the PeMADS proteins are hydrophilic in nature (File S1). The globularity of proteins was also evaluated and it was found that most of them were predicted to be globular except for PeMADS22. None of the proteins harboured a signal peptide sequence. Except for few PeMADS proteins, all proteins were predicted to be localised in nucleus (File S1). Two proteins, PeMADS37 and PeMADS25 were predicted to be plasma membrane localised and displayed a transmembrane helix region, while PeMADS54 (Mγ), PeMADS36 (OsMADS32-like) and PeMADS22 (A-class) were localised in mitochondria, cytoplasm and extracellular region, respectively.

Protein-protein interaction (PPI)

The putative physical interaction network among ABCDE classes of MADS-box proteins confirmed their role in development of specific floral whorls. The constructed interaction network amongst various proteins of classes A, B, C, D and E (Fig. 7) showed direct interaction with each other. The class A proteins (PeMADS20/21) showed interaction with other classes of MADS-box protein. With respect to interactions between members of class A and B, PeMADS21 of class A displayed interaction with all B class proteins (PeMADS2/3/4/5/6), while the PeMADS20 of A class showed interaction only with PeMADS3/4/5 (Fig. 7). A and B class proteins interact to regulate the development of petals. Interestingly, class A proteins showed interactions with proteins of class C and class D as well of the C class proteins, only PeMADS24 showed interaction with both the A class proteins (PeMADS20/21), while PeMADS25 did not show any interaction. Both the A class proteins (PeMADS20/21) showed interaction with D class protein (PeMADS38) (Fig. 7). Notable interactions were observed among class A and E proteins, and this can be correlated to their combined expression during sepal development. Both A class proteins showed to interact only with two E class (PeMADS12/13) proteins (Fig. 7). Intra-class interaction between both A class proteins was also observed. Within B class and C class proteins, PeMADS3/4/5 of B class, showed interaction with only PeMADS24 of C class but not with PeMADS25 (Fig. 7). PeMADS2/6 of B class did not show interaction with C class members. All the B class proteins show interaction with all E class proteins (PeMADS10/12/13), except PeMADS11 (Fig. 7). The C class and D class interaction was restricted with interaction of PeMADS24 (C class) with PeMADS38 (D class) (Fig. 7). Considering C class interaction with E class, all the C class genes interacted with three E class proteins (PeMADS10/12/13), but not with PeMADS11. E class proteins, PeMADS10/11/13, also showed interaction with each other (Fig. 7).

Fig. 7.

Protein-Protein Interaction (PPI) Network among MADS-box proteins of ABCDE classes


Duplication events and ortholog prediction

Nine duplication events (DEs) were predicted, out of which only one (PeMADS8-PeMADS11) was from type II, and the rest were from type I genes. In Mα type, three DEs (PeMADS48-PeMADS50, PeMADS47-PeMADS53 and PeMADS40-PeMADS39) and in Mγ type, five DEs (PeMADS60-PeMADS58, PeMADS33-PeMADS55, PeMADS33-PeMADS56, PeMADS55-PeMADS56, and PeMADS34-PeMADS30) were predicted (File S2). The orthologous MADS-box genes were predicted from A. thaliana and O. sativa. A total of 29 and 21 orthologous genes were found in A. thaliana and O. sativa, respectively (File S3).

Promoter analysis

The identification of cis-acting promoter elements disclosed the significance of MADS-box genes in growth and developmental processes. Several elements that direct spatio-temporal expression of genes identified showed correlation with the expression patterns of the concerned genes (File S4). Type I gene promoters predominantly carried elements (ACGTABOX, ACGTCBOX, ABRELATERD1, SORLIP1AT, SEF3MOTIFGM, SEF1MOTIF, SEF4MOTIFGM7S, AGAMOUSATCONSENSUS, AGATCONSENSUS) that are responsible for gynoecium specific expression. The promoter region of PeMADS39 gene showed presence of root and flower specific elements (ASF1MOTIFCAMV, CCAATBOX1, and CIACADIANLELHC). The Mγ gene promoters carried elements for flower specific expression (CCAATBOX1, MYB26PS, LEAFYATAG, and WUSATAG). The PeMADS54 gene harboured leaf specific expression element (CCA1ATLHCB1, DOFCOREZM) in its promoter region.

A similar trend was observed in promoter elements distribution in type II MADS-box genes as expected. The A-class MADS-box genes mainly carried elements (CCAATBOX1, AGL2ATCONSENSUS, CARGCW8GAT, AGATCONSENSUS, WUSATAG, and CCAATBOX1), that directs expression in sepal, and elements that are responsible for high expression in gynostemium (such as ACGTABOX, ACGTCBOX, ABRELATERD1, BOXIIPCCHS, GADOWNAT, SORLIP1AT, SORLIP5AT, SEF3MOTIFGM, SEF1MOTIF, and SEF4MOTIFGM7S). The PeMADS20 gene carried flower specific promoter elements (CARGCW8GAT, CARGNCAT, CCAATBOX1, CCAATBOX1, and MYB26PS). The PeMADS21/22 genes displayed root specific element (ASF1MOTIFCAMV).

The B-Class genes, with significant expression in reproductive tissues (flower, sepal, and petal), carried a combination of regulatory elements (CCAATBOX1, MYB26PS, AGL2ATCONSENSUS, and CARGCW8GAT) that have direct role in reproductive tissues. The C/D-class genes with role in female reproductive organ development harboured regulatory elements that are essential for expression in gynostemium (AGAMOUSATCONSENSUS, AGATCONSENSUS, and LEAFYATAG). Promoters of the E-class genes that participate in coordination of flower development showed predominant presence of flower development specific elements such as CCAATBOX1, and CARGCW8GAT. The other type II genes carried diverse elements in upstream region. The PeMADS36 (OsMADS32-type) gene promoter had root and leaf specific elements ASF1MOTIFCAMV, AGL1ATCONSENSUS, AGL3ATCONSENSUS, while the PeMADS28 (Bs-type) promoter carried flower and gynostemium specific elements (CCAATBOX1, ACGTCBOX, ABRELATERD1, BOXIIPCCHS, GADOWNAT, SEF3MOTIFGM, AGAMOUSATCONSENSUS, AGATCONSENSUS). The PeMADS23 gene showed expression in leaf, root, and floral stalk, and carried elements such as ASF1MOTIFCAMV, CCAATBOX1, CARGNCAT, AGL3ATCONSENSUS, LEAFYATAG, and CARGCW8GAT. The ANR1 (PeMAD27/26) and SOC1 type (PeMADS18/19) genes had leaf and root specific promoter elements (ASF1MOTIFCAMV, LEAFYATAG) (File S4).

Expression analysis

The expression profile of PeMADS genes in different tissues such as leaf, root, floral stalk, flower, sepal, petal, labellum and gynostemium was analysed on the basis of RPKM value. Genes having null RPKM values were not considered for analysis. The remaining 14 type I genes had high expression in gynostemium. Apart from gynostemium expression, the Mα genes, PeMADS37 expressed in floral stalk and floral parts, while PeMADS40 showed expression in flower. Interestingly, PeMADS39 exhibited expression in root as well as in flower. Similarly, in addition to gynostemium, majority of the Mγ genes displayed significant expression in flower and petal; PeMADS54 had high expression in leaf as well (Fig. 8A).

The three A class genes (PeMADS22/21/20) had high expression in gynostemium. The genes PeMADS22/21 had least expression in sepals and high expression in root, with PeMADS21 also expressing moderately in leaf (Fig. 8B). The five B class genes (PeMADS6/5/2/3/4) had least expression in vegetative parts and high expression in flower. Additionally, the PeMADS4 gene expressed in gynostemium, labellum and petal, while the PeMADS6/5/2 genes showed maximum expression in sepals and petals, and the PeMADS3 gene showed high expression in petal and labellum (Fig. 8). Of the two C class genes (PeMADS24/25), PeMADS24 gene showed expression in root, flower, sepal and gynostemium, and the PeMADS25 gene had expression in flower, petal and gynostemium (Fig. 8). Similarly, the two D genes (PeMADS38/7) had intense expression in gynostemium and petals. The PeMADS38 gene also had maximum expression in flower. The five E class genes (PeMADS13/11/8/12/10) had maximum expression in flower, sepal and petal. The PeMADS13/11 genes also showed maximum expression in labellum, and the PeMADS12/10 genes also showed moderate expression in labellum and gynostemium (Fig. 8).

The only SVP sub-family gene (PeMADS23) showed high expression in leaf, root and floral stalk. The Bs sub-family gene (PeMADS28) showed maximum expression in flower and gynostemium and moderate expression in petal. The ANR1 sub-family genes (PeMADS26/27) and SOC1 sub-family genes (PeMADS19/18) showed maximum expression in root (Fig. 8). The AGL6-like sub-family genes (PeMADS16/15) had maximum expression in sepal and petal and moderate expression in labellum and gynostemium. The single OsMADS32-like sub-family gene (PeMADS36) gene had expression in leaf, root and weak expression in flower. The only MIKC* type II gene (PeMADS9) had maximum expression in flower, petal and gynostemium (Fig. 8)

Discussion

The MADS-box genes are floral homeotic genes with critical role in plant growth and development and form the basis of ABCDE model of flowering. The highly complicated and advanced floral organization of Phalaenopsis equestris makes it an ideal organism to study florigenesis and the interrelationship of MADS-box genes in the process. In the present study, MADS-box (PeMADS) gene family is characterized in P. equestris extensively for various genomic and proteomic attributes. The size of MADS-box gene family varied widely across different plant species. The P. equestris genome carried more of type II MADS-box genes than type I MADS-box genes. A similar trend was reported with closely related genomes Apostasia shenzhenica (Zhang et al. 2017) and Dendrobium catenatum (Zhang et al. 2016) and in other plant genomes such as Glycine max (Fan et al. 2013), Brachypodium distachyon (Wei et al. 2014) and Malus domestica (Tian et al. 2015) as well (Table 2). This indicates, presumably, that the type II group members are evolutionarily more advanced than the type I group members (Lin et al. 2016). However, Arabidopsis thaliana consisted of more type I than the type II MADS-box genes. This can be attributed to unrelated gene duplication pattern of type I and type II groups (Parenicova et al. 2003), indicating different evolutionary strain in different plant groups. Such duplication events bring gain and loss of function events and has been proposed as an important mechanism for neo/ non-functionalization and sub-functionalization (Irish and Litt 2005). The lesser number of intron or intron-less genes in type 1 can be due to the simple gene structure and reverse transcribed organization (Parenicova et al. 2003; Wells et al. 2015). While the complex gene structure and homo- and hetero- dimer formation for specifying the floral organ identity have resulted in the more number of introns in type II genes (Theissen 2001; Egea-Cortines et al. 1999; Eckardt 2003). This further corroborates that the type II genes are evolutionary more advanced than the type I genes (Lin et al. 2016). The intron phase analysis indicated the occurrence of maximum 0 phase followed by 2 and 1 phase, suggesting the conserved nature of splicing phases in the MADS-box genes (Fig. 1A-B). The physico-chemical characters of PeMADS were also comparable in accordance with studies in Erycina pusilla (Lin et al. 2016), A. thaliana (Parenicova et al. 2003), Oryza sativa (Arora et al. 2007) and G. max (Shu et al. 2013).

Distribution of Type 1 and Type II genes in different plant genome

Type / PlantP. equestrisA. shenzhenica (Zhang et al. 2017)D. officinale Lindl. (Zhang et al. 2016)A. thaliana (Parenicova et al. 2003)O. sativa (Arora et al. 2007)Z. mays (Zhao et al. 2011)B. distachyon (Wei et al. 2014)G. max (Shu et al. 2013)M. domestica (Tian et al. 2015)S. bicolor (Zhao et al. 2011)T. aestivum (Ma et al. 2017)
16.66%13.8%23.8018.69%17.33%36%15.78%22.69%15.06%40%17.77%
---15.88%12%4%12.2%8.58%5.47%3.07%
25%11.11%20.63%19.62%13.33%2.66%3.50%14.72%16.43%3.07%2.77%
---3.73%----6.84%2.77%
Total Type I41.66%25%44.44%57.92%42.66%42.66%31.48%46%43.8%46.1%23.33%
MIKC56.25%69.44%50.79%40.18%52%52%56.14%49.69%51.36%50.76%76.66%
MIKC*2.08%5.55%4.76%1.86%5.53%5.53%12.28%4.29%4.79%3.07%
Total Type II58.3375%55%42%57.53%57.53%68.42%54%56.15%53.8476.66%
Total MADS-box genes48366310775755716314665180

In P. equestris, type I MADS-box proteins were grouped into Mα and Mγ. Interestingly, in E. pusilla only one group i.e. Mγ type is present (Lin et al. 2016), while in B. distachyon and A. thaliana, Mα, Mβ and Mγ groups are present (Wei et al. 2014; Parenicova et al. 2003) and all the four groups (Mα, Mβ, Mγ, and Mδ) are reported in Cucumis sativus (Hu and Liu, 2012). This inconsistency within the type I groups in different plant species indicate the varying birth and death rate of type I genes (Nam et al. 2004). Among type II MADS-box proteins, nine subgroups were formed in P. equestris (Cai et al. 2015), while nine in E. pusilla (Lin et al. 2016) and B. distachyon (Wei et al. 2014), 10 in C. sativus (Hu and Liu 2012), eight in M. domestica (Tian et al. 2015), 14 in O. sativa (Arora et al. 2007) and 13 were reported in Vitis vinifera (Diaz-Riquelme et al. 2009). The variability in number of subgroups in different plant species might be due to the requirement of specific functions of these groups (Shimeld 1999). Interestingly, the FLC genes involved in vernalization (Michaels and Amasino 1999) and the AGL12-like genes with a role in root proliferation and floral transition (Tapia-Lopez et al. 2008) were absent in P. equestris (Cai et al. 2015). But it is also reported that FLC are difficult to isolate owing to their selectively smaller size and highly divergent nature (Rouse et al. 2002). FLC and AGL12-like proteins probably have a different mechanism from proteins belonging to ANR1 and SOC1 (or AGL20) subgroups which are also responsible for root proliferation and floral transition (Hepworth et al. 2002; Liu et al. 2008; Zhang et al. 2000). The functioning of these genes in P. equestris might have been lost or diverged into a separate group during the course of evolution.

Reports on type I genes are sporadic and mostly focussed towards elucidating their role in plant reproduction and developmental stages, chiefly in female gametophyte, embryo and endosperm development (Bemer et al. 2008; Bouyer et al. 2011; Kang et al. 2008; Masiero et al.2011; Portereiko et al. 2006). PeMADS type I genes expressed predominantly in gynostemium with overlapping expression in sepal, labellum and flower (Fig. 8a) which is similar to earlier reports from B. distachyon (Wei et al. 2014), E. pusilla (Lin et al. 2016) and A. thaliana (Parenicova et al. 2003), suggesting their role in female gametophyte development. The Mα gene, PeMADS39, showed high expression in root and flower which is in line with the expression of Raphanus sativus genes (RsMADS093 and RsMADS111; Li et al. 2016) and C. sativus genes (CsMADS34/35; Hu and Liu 2012). The Mγ PeMADS54 is highly expressed in leaf, which is comparable with expression of PpeMADS31 of Prunus persica (Wells et al. 2015) and CsMADS36/37/38 in C. Sativus (Hu and Liu 2012). Additionally, this gene also has leaf specific elements in its promoter region (CCA1ATLHCB1, DOFCOREZM).

The type II genes clearly diverged into separate groups pertaining to A, B, C, D and E classes (Fig. 6). Malcomber and Kellogg (2005) suggests the evolution of A and E class proteins involved gene duplication events, and they coordinate with each other for sepal development (Coen and Meyerowitz 1991). Predicted interaction network projects intense interaction within A and E class members (Fig. 7). The expression profile also confirms that class A and E genes predominantly express in similar tissues (Fig. 8B). The expression of A class genes in gynostemium is also justified by the presence of gynostemium specific elements (ACGTABOX, ACGTCBOX, ABRELATERD1, BOXIIPCCHS, GADOWNAT, SORLIP1AT, SORLIP5AT, SEF3MOTIFGM, SEF1MOTIF, and SEF4MOTIFGM7S). Expression of A class genes in reproductive tissues was also been reported in Dendrobium (DoMADS2; Aceto and Gaudio 2011) and E. pusilla (EpMADS12; Lin et al. 2016). All genes of E class were highly expressed in sepals (Fig. 8B) which is similar to the expression of AdOM1 in Aranda Deborah (Lu et al. 1993) and EpMADS9 in E. pusilla (Lin et al. 2016). The putative interaction network also suggested that class A and E genes are associated with each other (Fig. 7) supporting their synergistic role for whorl 1 development (Pelaz et al. 2000). In addition, PeMADS21 and PeMADS22, the class A genes had root specific promoter element (ASF1MOTIFCAMV) and showed high expression in root tissues, similar to OsMADS18, a class A gene in O. sativa root (Fornara et al. 2004) indicating their additional role in root development.

The combined role of class A, B and E genes in whorl 2 development can be reflected in interaction studies (Fig. 7) which is in accordance with earlier reports (Pelaz et al. 2000). PeMADS22 (class A) gene had high expression in petals (Fig. 8B), similar to expression of DOMADS1 in Dendrobium (Yu and Goh 2000) and RsMADS68 in R. sativus (Li et al. 2016). All the genes of class B exhibited high expression in flower, similar to their expression in C. sativus (CsMADS21/22/23) (Hu and Liu 2012). The expression of PeMADS2/5/6 (class B) genes in petals was in conformity with class B genes of E. pusilla (EpMADS14/15/16) (Lin et al. 2016) and B. distachyon (BdMADS5) (Wei et al. 2014). All genes of class E of P. equestris were found to be upregulated in petals which is in concordance with expression of DcSEP1 in Dendrobium crumenatum (Xu et al. 2006), PhaMADS7 in Phalaenopsis ‘Athens’ (Acri-Nunes-Miranda and Mondragón-Palomino 2014) and RsMADS033 in R. sativus (Li et al. 2016). Furthermore, the remaining two class B genes, PeMADS3 and PeMADS4 expressed in labellum, as also reported for genes in E. pusilla (EpMADS13/14/16) (Lin et al. 2016) and Oncidium Gower Ramsey (OMADS8) (Chang et al. 2010). The expression of PeMADS4 gene in gynostemium tissue (Fig. 8C), can be related to the expression of BdMADS20 gene in carpel of B. distachyon (Wei et al. 2014).

Studies on P. equestris peloric mutants along with wild type by Tsai et al. (2004) revealed roles of specific genes in specific organ development. The sepals of peloric mutant had expression of only PeMADS2 in place of PeMADS2 and PeMADS5 for wild type flower, confirming critical role of PeMADS5 in sepal development. The expression of all four AP3-like genes were expressed in petals of wild flower except PeMADS4. In case of peloric mutant, PeMADS5 was not detected (lip-like petals), suggesting the role of PeMADS5 in petal formation. Moreover, PeMADS4 was highly expressed in labellum and gynostemium of wild flower. The expression of gene was noted in lip-like petals of peloric mutant flower, signifying the role of PeMADS4 in labellum formation. Furthermore, PeMADS3 gene have similar expression pattern in both wild and peloric mutant flower, indicating their role in inner perianth whorl. The PI-like gene (PeMADS6), found to be having no significant role in peloric mutant phenotype (Chen et al. 2012).

Many models have been proposed for understanding the functioning of these B class genes. Mondragon-Palomino and Theißen, (2008, 2009) proposed a model called ‘Orchid Code’ which implicit that combination of different genes to specify the different organ development. This model includes four different clades of DEF-like genes, which comes from the duplication of four DEF-like genes. Each specific clade expressed in specific organ of perianth. Clade 1 and 2 involve PeMADS2-like and OMADS3-like genes, respectively, which are present in all the tepals (Mondrago-Palomino and Theißen 2011), while clade 3 (PeMADS3-like) specifically expressed in inner tepals and clade 4 (PeMADS4-like) entirely shows expression in labellum. This Orchid Code described the expression of clade 1 and 2 in the outer tepals of flower and clade 1, 2 and 3 show presence in lateral inner tepals while clade 1, 2, 3 and 4 stipulate the formation of lip. Later, the Orchid Code was refined by Mondrago Palomino and Theißen (2011). The model includes the comparison of expression of four DEF-like genes in outer tepals, inner lateral tepals, labellum, gynostemium and ovary of different species (Vanilla planifolia, Phragmipedium longifolium, wild-type and peloric flowers of Phalaenopsis hybrid ‘Athens). Hsu et al. (2015) proposed another model named ‘Perianth or P-code’ based on the expression, specifying the perianth formation in Oncidium species. It involves the interaction of OPI with AP3/AGL6 that lead to the formation of two protein complexes, SP (sepal/petal) and L (Lip). The OPI gene is universally expressed in perianth. The sepal/petal development is determined by SP complex with OAP3-1 and OAGL6-1 proteins, while the L complex with OAP3-2 and OAGL6-2 determines the lip development. Together the negative interaction of SP and L complex governs the non-reproductive part of flower. This model is specifically applicable to perianth development, not for stamen and carpel formation.

The androecium (whorl 3) and gynoecium (whorl 4) in orchids are fused to form gynostemium. Theoretically, whorl 3 is controlled by Class B, C, and E. PeMADS4 (class B) gene and PeMADS24/25 (class C) genes had high expression in gynostemium and was comparable with class B genes PpeMADS56 of P. persica (Wells et al. 2015) and RsMADS078 of R. sativus (Li et al. 2016) in stamens, and with class C genes CeMADS1 of Cymbidium ensifoliumin gynostemium (Wang et al. 2011), RsMADS036 in R. sativus stamens (Li et al. 2016) and OMADS4 in Oncidium Gower Ramsey column (Hsu et al. 2010). Chen et al. (2012) reported that, PeMADS1 ectopic expression mutants turned petals to gynostemium-like structures. For E class genes PeMADS10/12 were expressed in gynostemium as similar to EpMADS6 gene of E. pusilla (Lin et al. 2016), SEP genes of A. thaliana (Pelaz et al. 2000) and EgMADS3 of Eucalyptus (Southerton et al. 1998). Conventionally, whorl 4 includes class C, D and E genes. Class C/D genes (PeMADS7/38/24/25) were highly expressed in gynostemium (Fig. 8B). This class genes, EpMADS20/21/22 of E. pusilla (Lin et al. 2016), PhaMADS8/10 of Phalaenopsis ‘Athens’ (Acri-Nunes-Miranda and Mondragón-Palomino 2014), PhalAG1 of Phalaenopsis Hatsuyuki (Song et al. 2006) and DthyrAG1 of D. thyrsiflorum (Skipper et al. 2006) were reported to have similar expression. The C class genes are also reported to have a role in fruit formation of P. persica (PpeMADS11/24/45) (Wells et al. 2015). Class D genes (PeMADS38, PeMADS7) were expressed in gynostemium, thus resembling the expression pattern of other class D genes of RsMADS047 in R. sativus (Li et al. 2016), BdMAD2 in B. distachyon (Wei et al. 2014) and EpMADS23 in E. pusilla (Lin et al. 2016). The E class genes (PeMADS12 and PeMADS10) have comparable expression pattern with VvMADS5 of V. vinifera (Boss et al. 2002). The control of whorl 3 and whorl 4 by class B, C, D and E genes, has also been revealed by the significant interaction among these genes on the basis of predicted protein structure (Fig. 7).

Class E genes (PeMADS8/10/11/12/13) were expressed in all the floral organs (Fig. 8B-E) which is in accordance with earlier reports on class E genes [RsMADS017/023 in R. sativus (Li et al. 2016), BdMADS11/26 in B. distachyon, EpMADS6 in E. pusilla (Lin et al. 2016) and OMADS6/11 in Oncidium Gower Ramsey (Chang et al. 2009)]. These results suggest that class E genes are required for the functioning of all floral whorls.

A significant interaction between class A and C genes was observed in the protein-protein interaction network (Fig. 7). This interaction has been reported to be quite significant but mutually antagonistic (Drews et al. 1991; Pelaz et al. 2000). Their mechanism of action can be explained on the basis of a number of studies. Class A genes are reported to be restricted to outer two whorls and prevent the activity of class C genes (Irish and Sussex 1990). Activity of class C genes, however, is reported in the inner two whorls restricting the expression of class A genes (Yanofsky et al. 1990). This is evident in their expression pattern (Fig. 8b). Interestingly, class A comprises of AP1 and AP2 genes, but the AP2 gene does not encode typical MADS-box domain (Riechmann and Meyerowitz 1997). The normal development of whorl 1 and 2 requires a significant expression of AP2 to prevent the accretion of AG transcripts, which could in turn trigger the formation of carpels instead (Drews et al. 1991; Jofuku et al. 1994). Similarly, the activity of class C genes is essential for floral determinacy; in the absence of which, there is a formation of indeterminate number of floral whorls (Pelaz et al. 2000). The antagonist interaction between class A and C was also reported in the ap2 mutant of A. thaliana (Coen and Meyerowitz 1991). Ectopic expression of a class C gene (PeMADS1) was reported to be associated with development of gynostemium-like petal in P. equestris (Chen et al. 2012).

The SVP gene subfamily in C. sativus (Hu and Liu 2012) and O. sativa (Arora et al. 2007) had crucial role in leaf and root development. The presence of leaf specific elements (LEAFYATAG) and expression of PeMADS23 reflects the same (Fig. 8F). The B sister gene EpMADS24 of E. pusilla (Lin et al. 2016) and VvBS2 of V. vinifera (Diaz-Riquelme et al. 2009) proposed to have a crucial role in cell differentiation during ovule and seed development, and similar expression was observed with PeMADS28 (B sister) gene in P. equestris. The SOC1 (or AGL20) subfamily genes (PeMADS18/19) were expressed in root and carried root and leaf specific promoter elements (ASF1MOTIFCAMV, LEAFYATAG) and the expression was similar to AGL14/19 of A. thaliana (Parenicova et al. 2003), and PpeMADS64 of P. persica (Wells et al. 2015). This up-regulation in root might also be associated with nutrient deficiency response (Gan et al. 2005). The role of SOC1 (or AGL20) genes in controlling flowering time has also been reported (Duan et al. 2015). In addition, PeMADS18 gene was also strongly detected in leaf as reported in V. vinifera (Diaz-Riquelme et al. 2009). The ANR1 subfamily (PeMADS26/27) was found to have similar expression pattern and similar promoter elements (ASF1MOTIFCAMV, LEAFYATAG) in root like CsMADS27/ 28/29 of C. sativus (Hu and Liu 2012) and AGL16/17/21 of A. thaliana (Parenicova et al. 2003), suggesting their function in lateral root growth in response to nutrients and root development (Gan et al. 2005; Zhang and forde 2000). The AGL6-like genes (PeMADS14/15/16) were expressed in sepal, petal and labellum (Fig. 8F), related to OsMADS6 of O. sativa, which known to regulate floral meristem determinacy and floral organ identity (Ohmori et al. 2009). OsMADS32-like gene has similar expression like OsMADS32 of O. sativa (Arora et al. 2007) and TaAGL14/15 in T. aestivum (Zhao et al. 2006) and probably have role in plant developmental processes. The MIKC* genes have similar expression pattern as in CsMADS30 in C. sativus (Hu and Liu 2012), but the functional significance of MIKC* is not much clear in development of floral whorls. The expression of MIKC* in gynostemium of P. equestris and preceding studies in A. thaliana (Parenicova et al. 2003), and E. pusilla (Lin et al. 2016) have suggested a role for MIKC* genes in gametophyte development.

Conclusions

In the present study, an attempt was made to validate ABCDE model in P. equestris, on the basis of structural and functional characterization of MADS-box genes with special thrust to expression profiling and protein-protein interaction. This provides a platform to functionally elucidate the mechanism of flower development and open newer vistas to functional validation of the MADS-box gene family in florigenesis.

Acknowledgements

Authors are grateful to Department of Science and Technology, Government of India for partial financial support under Promotion of University Research and Scientific Excellence (PURSE) grant scheme. JKS is thankful to Bio-CARe scheme (BT/Bio-CARe/647/2011-12) of Department of Biotechnology, Government of India, and Science and Engineering Research Board (SERB), Government of India for Early Career Research Award (ECR/2016/001190).

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  • Promotion of University Research and Scientific Excellence
     
     
  • Science and Engineering Research Board
      10.13039/501100001843
     
  • Bio-CARe scheme
     
      BT/Bio-CARe/647/2011-12
  • Early Career Research Award
     
      ECR/2016/001190
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