Insights into Genetics of Floral Development in Amborella trichopoda Baill through Genome-wide Survey and Expression Analysis of MADS-Box Transcription Factors

Insights into Genetics of Floral Development in Amborella trichopoda Baill through Genome-wide Survey and Expression Analysis of MADS-Box Transcription Factors | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Insights into Genetics of Floral Development in Amborella trichopoda Baill through Genome-wide Survey and Expression Analysis of MADS-Box Transcription Factors Sanam Parajuli, Bibek Adhikari, Madhav P. Nepal This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5314709/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 12 Feb, 2025 Read the published version in Scientific Reports → Version 1 posted 9 You are reading this latest preprint version Abstract The ABCDE model is a well-known general model of floral development in angiosperms with perfect flowers, with some modifications in different plant taxa. The Fading Borders Model was proposed to better explain floral patterning in basal angiosperms that typically possess spirally arranged floral organs. The MADS-Box gene family is central to these models and has greatly expanded in higher plants which is associated with increasing complexity in floral structures. Amborella trichopoda is a basal angiosperm with simpler floral features, and the genetic and functional roles of MADS-box genes in floral development remain poorly understood in the species. The major objectives of this study were to perform a genome-wide identification and characterization of MADS-BOX genes in A. trichopoda , and to analyze their expression in floral buds and mature flowers t. We identified 42 members of the MADS-Box gene family in A. trichopoda with a Hidden Markov Model (HMM)-based genome-wide survey. Among them, 27 were classified into Type-II or MIKC group. Based on our classification and orthology analysis, a direct ortholog APETALA1 ( AP1 ), an A-class floral MADS-Box gene was absent in A. trichopoda . Gene expression analysis indicated that MIKC-type genes were differentially expressed between male and female flowers with B-function orthologs: APETALA3 ( AP3 ) and PISTILLATA ( PI ) in the species having differential expression between the two sexes, and E-function orthologs being upregulated in female flowers. Based on these findings, we propose a modification in the Fading Borders Model in A. trichopoda with a modified A-function, B- and E-function orthologs’ expression being sex-specific, and C- and D-function genes having roles similar to that in the classical ABCDE model. These results provide new insights into the genetics underlying floral patterning in the basal angiosperms. Biological sciences/Evolution/Evolutionary genetics Biological sciences/Genetics/Development Biological sciences/Genetics/Evolutionary biology Biological sciences/Genetics/Gene expression Biological sciences/Genetics/Gene regulation Biological sciences/Plant sciences/Plant development Biological sciences/Plant sciences/Plant evolution Biological sciences/Plant sciences/Plant genetics Biological sciences/Plant sciences/Plant molecular biology Biological sciences/Plant sciences/Plant reproduction Biological sciences/Plant sciences/Plant signalling Biological sciences/Molecular biology/Transcriptomics Biological sciences/Evolution Biological sciences/Computational biology and bioinformatics Biological sciences/Computational biology and bioinformatics/Classification and taxonomy Biological sciences/Computational biology and bioinformatics/Gene ontology Biological sciences/Computational biology and bioinformatics/Phylogeny Biological sciences/Computational biology and bioinformatics/Protein analysis Biological sciences/Computational biology and bioinformatics/Protein function predictions Amborella trichopoda Basal Lineage of Angiosperms MADS-Box Transcription Factors Fading Borders Model Flowering Plants Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Introduction Plants undergo a vegetative to reproductive switch in their life cycle, and the onset of this switch is influenced by environmental cues, along with several endogenous developmental factors 1 . These signals are carefully timed for resources like pollinator activity, water availability, wind, etc. crucial for the plants’ reproductive success 2 . This transition replaces vegetative meristems with floral meristems, after which cells in the floral meristem differentiate into tissues that make up the floral whorls: calyx, corolla, androecium, and gynoecium in a perfect flower. The genes regulating this patterning were first identified in Antirrhinum majus 3 and Arabidopsis thaliana 4 with homeotic mutants, where one floral organ is replaced by another as a result of mutation(s) in key regulatory genes. With systematic analyses of these mutations, the ABC model of floral patterning was proposed in 1991 5 . This model explains that three classes of genes contribute to floral patterning with overlapping whorls of expression: sepal identity is determined by A-function genes (e.g., Arabidopsis APETALA1, AP1 and AP2 ), petals by A- and B- function genes (e.g., Arabidopsis AP3 and PISTILLATA , PI ), androecium by B- and C- function genes (e.g., Arabidopsis AGAMOUS , AG ), and gynoecium by C-function genes 5 . Later, the model was expanded to the ABCDE model after the discovery of D-function genes (e.g.: Arabidopsis AGAMOUS-LIKE11 , AGL11 ) that determine ovule identity 6 and E-function genes (e.g., Arabidopsis SEPALLATA1, SEP1 to SEP4 ) that acted in conjunction with B- and C-function genes in their respective whorls 7 . All floral identity and patterning genes, except A. thaliana AP2 and its orthologs, have a highly conserved domain known as the MADS-Domain and are collectively called floral MADS-Box genes 8 . The abbreviation MADS refers to a group of genes from different organisms: MINICHROMOSOME MAINTENANCE1 ( MCM1 ) from yeast, AGAMOUS ( AG ) from Arabidopsis , DEFICIENS ( DEF ) from Antirrhinum and Serum Response Factor ( SRF ) from humans, all encoding for proteins with an approximately 60 amino acid-long DNA-binding MADS-Domain in eukaryotes 9 . Genes containing the MADS or the M-domain collectively form the MADS-Box gene family. Proteins encoded by these genes are transcription factors that bind to promoter regions with a highly conserved nucleotide motif called the CArG-box (C-A-rich-G-box) with the consensus sequence 5’-CC(A/T) 6 GG-3’, or other similar sites like the “N-10 type CArG box” or the Monocyte Enhancer Factor2 (MEF2) consensus binding site 10 . The dependence of floral identity on MADS-Box homeotic genes clearly puts duplication and diversification of these genes at the center of flower evolution 11 . Based on structural differences, MADS-Box proteins are broadly divided into two types: Type I and Type II. Type I genes are also called the M-type genes and Type II are known as MIKC-type, owing to their structure that contains the MADS (M), Intervening (I), Keratin-like (K), and C-terminal (C) domains 12 . Most Type I genes have a single exon and lack the K-box 10,13,14 . The K-box is an approximately 70 amino acid-long domain unique to plant’s MIKC-type MADS-Box proteins 10 and is believed to have evolved in the extant Streptophytes after the divergence from the common ancestor of plants and animals 700 million years ago (MYA) 15 . The Type-I genes are further classified as Ma, Mb, and Mg based on their phylogenetic nesting 16 , while Type-II genes are divided into MIKC C and MIKC*-types based on intron-exon structures, a longer I-domain, and a K-domain encoded by more exons in the MIKC*-type 12 , also known as Mδ genes 16 . Represented by a single MIKC-type gene in the Charophycean alga Chara globularis 17 , MADS-Box genes have greatly expanded in higher plants. Amborella trichopoda Baill., the sole member of the family Amborellaceae, is considered the sister species to all extant angiosperms, and hence has caught the attention of botanists and evolutionary biologists worldwide. Native to New Caledonia, this woody evergreen shrub has been the focus of numerous prominent evolutionary studies 18–20 . Amborella is a dioecious species. meaning individual plants bear either male or female flowers arranged in botryoids panicles that are poorly branched. Male flowers measuring 4-5mm are slightly larger than female flowers measuring 3-4mm in diameter, and both are creamy white in color, with all floral organs spirally arranged. Each flower is surrounded by two prophylls, followed by spirally arranged tepals (9–11 in males, 7–8 in females), forming the perianth. The number of stamens in the male flower can range from 12–21, some inner ones in the spiral occasionally being sterile. Female flowers typically have five carpels and usually contain one or two staminodes, or sterile stamens, which still possess pollen sacs. However, pollen is not formed in female flowers as the pollen development ceases just before meiosis. Male flowers may also have an undifferentiated bulge in the center of the flower that is sometimes described as a rudimentary carpel 21 . Because the ABCDE model was based on observations on perfect flowers of higher angiosperms like Antirrhinum , Arabidopsis , and Petunia , it does not fully explain floral patterning in plants with simpler and unisexual flowers. In the case of Amborella trichopoda , flower organs are spirally arranged, transitioning from bracts to tepals and stamens or carpels. The “Fading Borders” model of floral patterning was proposed in 2004 for Amborella and other basal angiosperms, suggesting that floral organ identity genes are broadly expressed in the floral meristem but weakly expressed in the outer and inner edges 22 . Subsequent analyses revealed that AP1 (A-function) homologs are expressed in all floral organs and leaves, AP3/PI (B-function) homologs are expressed in all floral organs, and AG (C-function) homologs of basal angiosperms follow the classical ABC model in being expressed in both stamens and carpels 23 . However, the fading borders model does not explain the mechanism in dioecious plants like Amborella , nor how the floral patterning genes function in unisexual flowers. The model was suggested based on expression data, which might not capture genes that are unexpressed or get expressed at undetectable levels. A scaffold-level A. trichopoda genome was released in 2013 and 36 MADS-Box genes were predicted 24 . However, the identified genes were not characterized in detail for structure and expression, although protein-protein interactions among them were analyzed with hybrid assays. A recent study identified several Amborella genes associated with male gametophyte development, which included MIKC-type MADS-Box genes 25 . MIKC C -type genes in Amborella were identified in a more recent study, but identification of Type-I genes was outside its scope, and the identified genes were not characterized in detail 26 . A thorough genome-wide analysis and characterization study for MADS-Box genes in A. trichopoda has not been conducted yet, despite the gene family’s immense importance in plant development functions. With the availability of a chromosome-level genome assembly, we now have an opportunity to explore the functional and evolutionary dynamics of these genes in this basal angiosperm. The primary objectives of this study were to conduct genome-wide identification and characterization of A. trichopoda MADS-Box genes, establish their orthologous relationships with A. thaliana MADS-Box genes, and analyze their expression in floral buds and mature flowers. This will help us elucidate the genetic mechanisms underlying floral transitions, floral patterning, and sex expression in A. trichopoda . Results Taxonomy and Phylogeny of A. trichopoda MADS-Box Genes We identified 42 MADS-Box containing genes in the A. trichopoda genome. Based on their phylogenetic relationships with A. thaliana MADS-Box genes, they were classified into all the major identified groups of MADS-Box genes: 20 gene members belonged to the MIKC C group, seven belonged to the MIKC*, eight belonged to Ma, three to Mb, and four to Mg group (Table 1 ). The sequences of the identified proteins sequences are available in Supplementary File S1 . We could resolve putative orthologies of 20 A. trichopoda genes to A. thaliana MADS-Box genes with maximum likelihood phylogenetics of the full-length proteins (Fig. 1 ) and reciprocal best hit (RBH), a sequence similarity-based method. As shown in Fig. 1 , MIKC C proteins are grouped into 13 subfamilies: AGL1(AG/SHP/STK), AGL16, AGL12, AGL7 (AP1/CAL/FUL), AGL6, AGL4/9 (SEP), AP3/PI, AGL70 (MAF/FYF), AGL20 (SOC1), AGL22 (SVP), AGL32, AGL15, and one subfamily with three Amborella proteins but no Arabidopsis protein, placed in a clade ancestral to the AGL12, CAL, AGL13, and SEP subfamilies. There were no Amborella proteins in the MAF subfamily. Interestingly, an entire subfamily of MIKC* genes present in the Amborella genome was absent in the Arabidopsis genome. All floral MADS-Box, A, B-, C- and E-function, orthologs were present in the A. trichopoda genome, represented by one AGL7/8/10/ 79 (AmtrAGL7/8/10/ 79), two APETALLA3 ( AmtrAP3-1 and − 2 ), two PISTILLATA (AmtrPI-1 and AmtrPI-2 ), one AGAMOUS ( AmtrAG ), and two SEPALLATA orthologs ( AmtrAGL4 and 9 ), respectively. Any close D-function orthologs ( AGL11 ), were not predicted by our methods, although AmTrH2.11G126900.1.p did nest in the AG/SHP/STK clade. Table 1 Classification and nomenclature of Amborella trichopoda MADS-box proteins based on orthologous relationships inferred from the Maximum Likelihood tree (Fig. 1 ) and the Reciprocal Best Hit (RBH) analysis ( Supplementary File S2 ) Sequences from A. trichopoda that lack a putative ortholog in Arabidopsis thaliana are designated as N/A. A. trichopoda Protein Closest A. thaliana Ortholog Ortholog-based Name Class AmTrH2.01G136300.1.p AG AmtrAG MIKC C AmTrH2.11G127800.1.p AGL12 AmtrAGL12 MIKC C AmTrH2.01G083000.1.p AGL15 AmtrAGL15 MIKC C AmTrH2.13G099300.1.p AGL16/44/17/21 AmtrAGL16/44/17/21 MIKC C AmTrH2.04G078400.1.p AGL22 AmtrAGL22 MIKC C AmTrH2.09G085100.1.p AGL32 AmtrAGL32 MIKC C AmTrH2.10G036200.1.p AGL4 AmtrAGL4 MIKC C AmTrH2.09G080800.1.p AGL6/13 AmtrAGL6/13 MIKC C AmTrH2.10G037100.1.p AGL7/8/10/79 AmtrAGL7/8/10/79 MIKC C AmTrH2.06G043000.1.p AGL9 AmtrAGL9 MIKC C AmTrH2.06G157000.1.p AP3 AmtrAP3-1 MIKC C AmTrH2.09G064400.1.p AP3 AmtrAP3-2 MIKC C AmTrH2.06G101000.1.p PI AmtrPI-1 MIKC C AmTrH2.11G096800.1.p PI AmtrPI-2 MIKC C AmTrH2.05G000900.1.p N/A N/A MIKC C AmTrH2.06G043400.1.p N/A N/A MIKC C AmTrH2.06G043600.1.p N/A N/A MIKC C AmTrH2.09G064500.1.p N/A N/A MIKC C AmTrH2.09G080700.1.p AGL20/42/71/72 AmtrAGL20/42/71/72 MIKC C AmTrH2.11G126900.1.p N/A N/A MIKC C AmTrH2.04G150100.1.p AGL66/67/104 AmtrAGL66/67/104 MIKC* AmTrH2.04G179300.1.p AGL30 AmtrAGL30 MIKC* AmTrH2.04G099600.1.p N/A N/A MIKC* AmTrH2.08G064100.1.p N/A N/A MIKC* AmTrH2.09G031900.1.p N/A N/A MIKC* AmTrH2.09G032000.1.p N/A N/A MIKC* AmTrH2.10G045800.1.p N/A N/A MIKC* AmTrH2.01G037700.1.p AGL80 AmtrAGL80 Mg AmTrH2.03G113500.1.p N/A N/A Mg AmTrH2.04G040100.1.p N/A N/A Mg AmTrH2.11G086700.1.p N/A N/A Mg AmTrH2.03G034800.1.p AGL103 AmtrAGL103 Mb AmTrH2.01G056400.1.p N/A N/A Mb AmTrH2.01G174800.1.p N/A N/A Mb AmTrH2.01G163400.1.p AGL62 AmtrAGL62 Ma AmTrH2.01G096900.1.p N/A N/A Ma AmTrH2.01G097000.1.p N/A N/A Ma AmTrH2.01G097100.1.p N/A N/A Ma AmTrH2.07G063700.1.p N/A N/A Ma AmTrH2.07G063800.1.p N/A N/A Ma AmTrH2.07G084400.1.p N/A N/A Ma AmTrH2.13G009700.1.p N/A N/A Ma Physicochemical Properties and Sub-Cellular Localization As shown in Table 2 , the longest and largest MADS-Box protein in A. trichopoda was predicted to be AmtrAGL103 (Mb class), with a length of 378 amino acids (aa) and molecular weight (MW) of 43318.49 Daltons (Da). It was followed by AmtrAGL66/67/104 (MIKC*) which was 359 aa long and had a MW of 40813.66 Da. The shortest MADS-Box protein was predicted to be AmTrH2.04G099600.1.p with 99aa in length and 11539.68 Da in MW. Only nine of 42 MADS-Box proteins were predicted to have acidic isoelectric points (pI < 6). Consistent with the nature of transcription factors, 33 of the 42 identified MADS-Box proteins were predicted to localize in the nucleus, while the remaining nine were chloroplast-localized, albeit with scores below 1. Table 2 , Summary of Predicted Physicochemical Properties and subcellular localization of A. trichopoda MADS-Box proteins. MADS-Box Protein Length (aa) Theoretical Isoelectric Points (pI) Molecular Weight (Daltons) Predicted Subcellular Localization AmtrAG 235 8.94 26978.62 Nucleus AmtrAGL12 204 7.09 23277.81 Nucleus AmtrAGL15 257 8.39 29120.7 Nucleus AmtrAGL16/44/17/21 273 9.59 31351.45 Nucleus AmtrAGL22 221 5.69 25144.54 Nucleus AmtrAGL32 231 8.38 26867.71 Nucleus AmtrAGL4 243 9.1 27832 Nucleus AmtrAGL6/13 266 8.98 30379.49 Nucleus AmtrAGL7/8/10/79 242 8.52 28349.11 Nucleus AmtrAGL9 241 8.58 27357 Nucleus AmtrAP3-1 220 8.54 26079.52 Nucleus AmtrAP3-2 221 8.96 26166.71 Nucleus AmtrPI-1 212 9.04 24277.68 Nucleus AmtrPI-2 211 9.35 24725.19 Chloroplast AmTrH2.05G000900.1.p 257 10.02 29519.19 Chloroplast AmTrH2.06G043400.1.p 188 9.47 22018.74 Nucleus AmTrH2.06G043600.1.p 216 9.49 25246.14 Chloroplast AmTrH2.09G064500.1.p 210 9.28 24276.93 Nucleus AmtrAGL20/42/71/72 243 9.36 27541.53 Nucleus AmTrH2.11G126900.1.p 222 9.21 25650 Nucleus AmtrAGL66/67/104 359 5.89 40813.66 Nucleus AmtrAGL30 347 7.25 39375.56 Nucleus AmTrH2.04G099600.1.p 99 9.56 11539.68 Chloroplast AmTrH2.08G064100.1.p 108 8.95 12947.24 Chloroplast AmTrH2.09G031900.1.p 304 6.1 34061.15 Nucleus AmTrH2.09G032000.1.p 186 7.8 22020.07 Chloroplast AmTrH2.10G045800.1.p 335 9.26 38133.14 Nucleus AmtrAGL80 228 9.51 25602.17 Nucleus AmTrH2.03G113500.1.p 106 9.48 12218.09 Chloroplast AmTrH2.04G040100.1.p 355 5.36 39804.9 Nucleus AmTrH2.11G086700.1.p 144 7.63 16633.06 Nucleus AmtrAGL103 378 6.56 43318.49 Nucleus AmTrH2.01G056400.1.p 199 5.52 22541.63 Nucleus AmTrH2.01G174800.1.p 328 7.63 38151.46 Nucleus AmtrAGL62 212 9.25 23550.16 Nucleus AmTrH2.01G096900.1.p 211 5.35 24405.3 Nucleus AmTrH2.01G097000.1.p 189 7.61 21539.7 Chloroplast AmTrH2.01G097100.1.p 211 8.56 23924.15 Chloroplast AmTrH2.07G063700.1.p 150 9.48 16873.24 Nucleus AmTrH2.07G063800.1.p 262 5.16 29351.93 Nucleus AmTrH2.07G084400.1.p 367 5.14 41216.45 Nucleus AmTrH2.13G009700.1.p 153 8.63 16793.49 Nucleus Conserved Motif Composition of A. trichopoda MADS-Box proteins Analysis of conserved motifs in A. trichopoda MADS-Box proteins showed distinct class-specific motif compositions as shown in Fig. 2 . Most MIKC-type proteins had similar motif composition in their M-domains (Motifs 2, 1, and 4), with some exceptions. All MIKC C -type proteins were characterized by the presence of the K-domain (Motif 3), while MIKC* proteins lacked the K-domain and had a slightly shorter motif signature (Motif 6) instead of the K-domain. All but one Mg protein (AmtrAGL80) lacked the K-domain, despite most of them having the 2, 1, and 4 motifs representing the M-domain. Motifs 5 and 7 were unique to Ma proteins, while Motif 8 was seen in Mg and Mb proteins, and interestingly, the two PI orthologs (AmtrPI-1 and − 2). Motif 10, which differs slightly from Motif 2, was unique to Mg proteins. Conserved Motif 9 was present in the C-terminal regions of the two SEPALLATA othologs (AmtrAGL-4 and − 9) and AmtrAGL6/13, proteins that nested in the same sub-family in our ML tree (Fig. 1 ). Structure Analysis of A. trichopoda MADS-Box Genes Structure analysis of MADS-Box genes in A. trichopoda revealed class-specific exon-intron composition patterns (see Fig. 3 ). MIKC* and MIKC C -type genes were characterized by multiple exons. The first exon in all Type-II MADS-Box genes corresponded to the one encoding the M-domain. AmTrH2.06G043600.1.p was the longest MADS-Box gene in A. trichopoda at 79,504 bp long, while the shortest one was AmTrH2.03G113500.1.p (504bp). The B-function gene orthologs ( AmtrAP3-1 and − 2 ; AmtrPI-1 and − 2 ) were among the shortest genes in the Type-II group, with AmTrH2.09G064500.1.p being the other AmtrAGL66/67/104 had the greatest number of exons (11), while many Type-I MADS-Box genes had a single exon. AmtrAGL103 ’s single exon was the longest in all MADS-Box genes (1137bp). Details of gene structural attributes are summarized in Supplementary File S3 . Cis-Regulatory Elements (CREs) of A. trichopoda MADS-Box Genes Altogether 104 distinct cis -regulatory elements were predicted in the 2000bp upstream regions of the 42 A. trichopoda MADS-Box genes. The co-ordinates of the 20 most prevalent CREs are shown in Fig. 5 . With an average occurrence per sequence of 44.90, TATA-Box was the most common cis-element, followed by CAAT-Box (35.33). The CTCC motif followed occurring at an average of 10.57 times per gene. A complete list of all identified cis -element in the 2000bp upstream regions of MADS-Box genes and their co-ordinates are summarized in Supplementary File S4 . Gene Ontology Gene Ontology analysis of the identified MADS-Box proteins returned 22 GO terms corresponding to their predicted functions, as visualized in Fig. 5 . Under the “Cellular Component” ontology, “Nucleus” had the greatest number of hits (35), “Molecular Function” ontology had “DNA-binding transcription factor activity, RNA polymerase II-specific”, and “protein dimerization activity” had 35 hits each. Under the “Biological Process” ontology, “regulation of transcription by RNA polymerase II” had the highest number of hits (20), followed by “positive regulation of transcription by RNA polymerase II” (16). All these assigned ontologies are consistent with MADS-Box proteins being transcription factors that form multimers and bind to DNA. Chromosomal Locations of MADS-Box Genes in A. trichopoda Figure 6 shows MADS-Box genes mapped to 11 of the 13 chromosomes of A. trichopoda , with none located on chromosomes 2 and 12. Chromosome 1 had the highest concentration of MADS-Box genes (nine in total) while chromosomes 5 and 8 had one each. All MADS-Box genes on chromosome 6 were MIKC C -type, while three of five MADS-Box genes on chromosome 4 were MIKC*-type. Chromosomes 9 and 10 exclusively contained MIKC-type genes while chromosome 7 contained only Ma genes. Evolutionary Selection Pressure in Amborella trichopoda MADS-Box genes (Ka/Ks analysis) A Ka/Ks analysis of A. trichopoda MADS-Box genes revealed an average Ka/Ks value of 0.308 among all possible gene pair combinations, suggesting a strong purifying selection among these genes. The only paralogous gene pairs with a Ka/Ks value of more than one (> 1) were AmTrH2.01G096900.1/AmTrH2.01G097000.1 and AmTrH2.01G097100.1/ AmTrH2.01G097000.1 with Ka/Ks values of 1.08 and 1.07 respectively. All these genes belong to the Type I (Ma) group. Ka/Ks values of all gene pairs are provided in Supplementary File S5 . Collinearity of A. trichopoda MADS-Box Genes with A. thaliana MADS-Box Genes Out of 85230 total genes in both species, 1790 (2.10%) formed collinear blocks (defined as five consecutive genes) between the genomes. As shown in Fig. 7 , four MADS-Box genes in Amborella were found in collinear blocks with Arabidopsis , two of which ( AmtrAGL30 and AmtrAGL66/67/104 ) were of the MIKC*-type, and were located on chromosome 4. Also, AmtrAGL7/8/10/79 was collinear with Arabidopsis AGL7 and AmtrAGL12 was collinear with its Arabidopsis ortholog. No Type-I MADS-Box genes were found in collinear blocks between the two species. Differential Expression of A. trichopoda MADS-Box Genes in Male and Female Tissues Twenty MADS-Box genes were found to be differentially expressed between mature male and female flowers of A. trichopoda (padj ≤ 0.05), and all of them were Type-II MADS-Box genes. Twelve of these genes were downregulated while eight were upregulated in females. AmtrPI-2 gene had the most significant downregulation in female flowers (padj = 1.64e-134 and log2FC = -2.82) while the MIKC* gene AmtrAGL66/67/104 had the highest downregulation in terms of log2FC value in females compared to males (padj = 2.55e-70 and log2FC = -7.48). Among the upregulated genes in mature female flowers, AmtrAGL32 had the highest upregulation (log2FC = 7.26, padj = 1.27e-27), followed by AmTrH2.11G126900.1 (log2FC = 4.09, padj = 1.84e-37). Two AP3 orthologs exhibited distinct expression pattern: AmtrAP3-1 was upregulated in female flowers while AmtrAP3-2 was upregulated in male flowers. The volcano plot in Fig. 8 summarizes the differential expression of MADS-Box genes in mature Amborella male and female flowers. Principal Component Analysis (PCA) of the normalized raw read counts in the male and female mature flowers showed a clear distinction between two sexes based on the expression of MADS-Box genes (Fig. 9 ) , suggesting a distinct sex-specific expression pattern of MADS-Box genes in mature flowers of Amborella . The heatmap in Fig. 10 shows expression patterns of MADS-Box genes with non-zero read counts in mature flowers of the two sexes with z-transformed Transcript per Million kilobase (TPM) values. The samples distinctly clustered according to their sex based on the expression patterns of MADS-Box genes in mature flowers. In floral buds, only seven MADS-Box genes were differentially expressed between male and female samples (Fig. 11 ) . Of them, four were downregulated in females: AmTrH2.11G126900.1 (log2FC = -1.05. padj = 7.46e-07). AmtrAGL32 (log2FC value: -4.51, padj = 0.0009) and AmtrAGL15 (log2FC = -0.55, padj = 0.0005) in female buds. The three upregulated genes were AmtrAP3-2 , AmtrPI-2 , AmTrH2.09G064500.1 , and AmTrH2.07G063800.1 . Interestingly, AmtrAP3-2 and AmtrPI-2 were downregulated in mature female flowers but are upregulated in female floral buds. Additionally, AmTrH2.07G063800.1 , an Ma gene, was the only Type-I MADS-Box gene differentially expressed between male and female floral buds. Principal Component Analysis (PCA) of normalized read counts of male and female floral buds had a lot of overlap between the 95% confidence interval ellipses (see Fig. 12 ), showing that unlike in mature flowers, the expression pattern of MADS-Box genes in floral buds of Amborella is not highly sex-specific. Clustering of samples based on expression patterns (z-transformed normalized read counts) based on Euclidean distances could not cluster male and female floral buds distinctly, as shown in Fig. 13 . All gene expression data are available in Supplementary File S6 . Discussion Amborella trichopoda MADS-Box Genes Identification and Nomenclature We identified 42 MADS-Box genes in A. trichopoda which belonged to all the major classes of plant MADS-Box genes. We used a homology-based approach to name the genes, referencing their putative orthology with A. thaliana MADS-Box genes. With the decreasing costs of Next-Generation Sequencing (NGS) technologies, there has been a dramatic increase in the number of plant genomes coming from research groups around the world. Public availability of such genomes has subsequently accelerated genome-wide identification studies of genes and gene families in plants, advancing our understanding of evolutionary processes, functional genomics, and plant adaptation to diverse environments. However, the procedure for naming genes in a gene family has not been consistent across different studies. For instance, the identified MADS-Box genes were named following their chromosomal locations in Zizania latifolia 27 . In some cases, no specific naming criteria were applied as in the case of Glycine max 28 , Oryza sativa 29 , and Malus domestica 30 . MADS-Box genes were named after their gene ids in increasing order in Sesamum indicum 31 , and in two orchids: Dendrobium officinale and Phalaenopsis equestris , the authors did not specify how the identified genes were named, even though the genes were assigned orthologies with their A. thaliana counterparts 32 . These inconsistencies can present challenges not only in identifying genes, but also in assigning functions to these genes, as genes with the same number may not have the same functional annotation across species. A more consistent approach, such as assigning gene names based on verified or predicted biological functions inferred from sequence homology, would help standardize gene naming and facilitate cross-species comparisons. . Such standardizations in nomenclature have been suggested for multiple animal gene families 33–36 . Efforts have been made to standardize nomenclature for plant gene families as well. Orthology-based nomenclature for plant WRKY genes was recommended by Mohanta et al. in 2016 37 . Naming guidelines have been proposed for rice WRKY 38 , A. trichopoda WRKY 39 , plant HKT 38 , and MAPK 40 gene families. In the present study, we named each Amborella trichopoda MADS-Box gene by following an orthology-based approach, where the first two letters of the gene name are derived from the genus and species names, followed by 'AGLx' (AGAMOUS-like and a number x), corresponding to the orthologous A. thaliana gene. For instance, 'AmTrAGL1' refers to the AGL1 ortholog in Amborella trichopoda . Since 'At' is already used to abbreviate Arabidopsis thaliana , we used 'AmTr' (from the first two letters of the genus and species) to abbreviate Amborella trichopoda .. Assigning orthologies based solely on sequence similarity, however, might lead to errors in functional annotation because of possible differences in expression patterns, for instance, AG and AGL1 have a very similar sequence structure but can have distinct expression patterns and functions 8 . Gene expression patterns, mutagenic and/or overexpression studies can thus provide the strongest evidence of orthology 9,41 . Although a detailed report on A. trichopoda MADS-Box genes has not been published yet, previous studies have reported varying numbers of Amborella MADS-Box genes: 36 in one study 24 and 33 in another 32 . With initial HMMER 42 searches, we obtained 46 unique hits in this study, four of which were filtered out because they lacked MADS domain, as confirmed by the Conserved Domain Database (CDD) 43 , Simple Modular Architecture Research Tool (SMART) 44 or Pfam 45 domain searches. In fact, The Arabidopsis Information Resource (TAIR) 46 ’s A. thaliana MADS-Box gene family entry from AGRIS had 109 gene entries at the time of writing this article, but three of those genes did not have the MADS domain as per our domain search and three did not have a predicted protein sequence, and hence were excluded from further analyses. Upon manual analysis of hits from our two HMMER searches, one with a Pfam HMM profile entry PF00319, and the other with an HMM profile generated from alignment of all A. thaliana MADS-Box protein sequences, we could observe that all entries filtered from our domain confirmation were identified as hits from the first HMMER search itself, making the second HMMER search redundant. With this, we recommend that, if available, a HMMER search with the PFam HMM profile in the Interpro database would be sufficient to identify gene family homologs in a genome for the MADS-Box gene family, provided that the expect threshold (e-value) is not too selective, as it could potentially filter out distant homologs. Classification and Evolutionary Dynamics of A. trichopoda MADS-Box Genes Among the 42 A. trichopoda MADS-Box genes identified in this study, 20 genes were classified as MIKC C , seven as MIKC*, eight as Ma, three as Mb, and four as Mg. Different numbers of MADS-Box genes have been reported in various algae and plant species. No MIKC C or MIKC* genes were found in green algae, but a single gene with a MEF2-like M-domain without I, K, or C domains was reported in in Chlamydomonas reinhardtii and C. merolae 15 . One MIKC type gene was reported in Charophycean algae Chara globularis, C. scutata , and C. peracerosum-strigosum-littorale complex 17 , while 26 MADS-Box genes (17 MIKC and 9 M-type) were found in the bryophyte Physcomitrella patens 47 , 19 (6 MIKC and 13 M-type) in the lycophyte Selaginella moellendorffii 48 , and 36 (35 MIKC and 1 M-type) in the fern Vandenboschia speciosa 49 . The presence of MIKC* genes in bryophytes suggests that at least one MIKC* gene was present in the common ancestor of bryophytes and seedless vascular plants, likely evolving from an MIKC C -type gene (reviewed in 47 ). Despite MIKC C genes being present in seedless plants, they are not orthologous to phanerogamic (gymnosperm or angiosperm) MIKC C genes, hinting at independent evolution of these genes in these plant lineages 12 . MIKC* genes, on the other hand, show considerable homology and conservation of function across seedless plants, gymnosperms and angiosperms (reviewed in 47 ). The number of MADS-Box genes varies greatly in gymnosperms ranging from three (all MIKC) in Taxus baccata to 367 (350 MIKC and 17 M-type) in Pinus taeda 50 . In our study, we classified A. trichopoda MIKC C genes into 12 subfamilies: AGL1(AG/SHP/STK), AGL16, AGL12, AGL7 (AP1/CAL/FUL), AGL6, AGL4/9 (SEP), AP3/PI, AGL20 (SOC1), AGL22 (SVP), AGL32, AGL15, and a clade ancestral to the AGL12, CAL, AGL13, and SEP subfamilies, specific to Amborella . The MAF/FLC and FYF clades represented in A. thaliana were however absent in A. trichopoda . Our results agree largely with that of a previous study that identified 14 MIKC C clades in gymnosperms and basal angiosperms: SVP, MADS32, AP3/PI, AGL32, AGL15, AG, ANR1, AGL12, SOC1, GMADS, FLC, AP1/FUL, AGL6 , and SEP 51 , except for GMADS, which is gymnosperm-specific; MADS32 , which is a clade with OsMADS32 from rice, a species not included in our study, and MAF/FLC : a group found only in Asteraceae 52,53 , the botanical family of A. thaliana . Our results are largely similar to a recent study that reported 13 subfamilies of Amborella MIKC C genes, and identified single orthologs in PI and AG groups 26 . Our results confirm a previous report that only a single copy of AP1/FUL/SOC1 genes is present in A. trichopoda 51 . Absent in gymnosperms, the AP1/FUL family first appeared in angiosperms 54 , diverging into two unique groups in monocots and three groups in eudicots 51 . The presence of only one gene (name) in the AP1/FUL family in A. trichopoda that shared orthology to AGL- 7, 10, 9 , and 79 in our study suggests that AP1 ( AGL7) -specific orthologs are probably not present in A. trichopoda and evolved later in the angiosperm lineage. This result aligns with a previous report that that euAP1 may have originated from a frameshift mutation in an ancient euFUL- or FUL-like gene 54 . This absence of a direct AGL7 ortholog and statistically similar expression of the ancestral ortholog in both male and female flowers, discussed later, suggests the possibility of this gene carrying out the A-function or its complete absence in A. trichopoda , possibly explaining the absence of differentiated calyx and corolla whorls in its flowers. Orthologs of AP2 , a non-MADS-Box A-function gene, were found in A. trichopoda (blast search, data not shown), however, AmtrAGL7/8/10/79 was one of the four MADS-Box genes in Amborella – present in a genomic collinear block with Arabidopsis , suggesting a strong conservation of function of these genes across plant lineages. In Arabidopsis thaliana , B-function is controlled by the AP3 and PI genes 55 . We found two orthologs for each in Amborella trichopoda : AmtrAP3-1, AmtrAP3 - 2, AmtrPI-1 and AmtrPI-2 . These genes were placed in A. trichopoda ’s AP3/PI group in a previous study 51 , but the two AP3 orthologs were not named as such. AP3 orthologs have been identified in gymnosperms: PrDGL in Pinus radiata 56 , GGM13 and GGM2 in Gnetum gnemon 56 , and DEFICIENS-AGAMOUS-LIKE (DAL) − 11, 12 , and 13 in Picea abies 57 . The B-function of specifying male reproductive organ identity is conserved in conifer DEF/GLO-like proteins, but in contrast, GGM13 -like genes is expressed preferentially in female tissues 50 , also known as B-sister MADS-Box genes 58 . Since AmtrAP3-1 is not related to GGM13 in our phylogenetic analysis, it is not a B-sister MADS-Box gene. Instead, one A. trichopoda protein AmTrH2.09G085100.1.p (AmtrAGL32) nested in the AGL32 ( Arabidopsis B-sister) clade in our tree. Expression data (discussed later) showed upregulation of this gene in mature female flowers, suggesting potential conservation of function of B-sister MADS-Box genes in early angiosperms and gymnosperms. Since GGM13 was already present in gymnosperms, we infer that the A. trichopoda B-sister gene is a descendant of gymnosperm B-sister MADS-Box genes and likely existed before the divergence of angiosperms and gymnosperms. The AmtrAP3-1 gene was longer than AmtrAP3-2 , but the proteins they encode were nearly equal lengths, with the gene length difference attributed to intron size. Both proteins had nearly identical sequences and were characterized by the “DLRLG” motif at the C-terminal end, a signature paleoAP3 motif found in AP3 proteins in lower angiosperms. This supports the idea that the paleoAP3 motif is the common ancestral form of the euAP3 and TM6 lineages found in higher eudicots. Duplication in a paleoAP3 ancestor gave rise to the new lineages, and this event occurred after the common ancestor of Buxaceae and higher eudicots, but not later than the diversification into higher eudicot classes 59 . A. trichopoda also has a distinct PI lineage, consisting of two PI orthologs. The PI lineage was probably formed in the lineage leading to angiosperms as a result of a duplication in the B-function gene lineage, of which elimination of the paleoAP3 motif was a major change in the new lineage 59 . AP3 and PI proteins share striking sequence similarity, except for the characteristic residues especially concentrated at the C-terminal motifs 60 . We are the first to report the presence of two PI orthologs in A. trichopoda genome. C-function floral MADS-Box genes specify stamen and carpel identity and are represented by AGAMOUS ( AG ), SHATTERPROOF 1 ( SHP1 / AGL1 ) and SHATTERPROOF 2 ( SHP2/AGL5 ), while D-function genes specify ovule identity, represented by AGL11 ( STK ) in A. thaliana (reviewed in 61 ). In A. trichopoda , the AG/SHP/STK clade had two genes: 1) AmTrH2.01G136300 , an A. thaliana AG ortholog, hence named AmtrAG and 2) AmTrH2.11G126900 nested in the AG/SHP/STK clade, and the fact that it was upregulated in female buds and mature female flowers, suggests that it may be a D-function ortholog. The expression of AmtrAG in both male and female flowers further supports our orthology assignment to A. thaliana AG gene. C-function MADS-Box gene orthologs have been found in all seed plants, but not in non-seed plants 61 . Members of the AGAMOUS clade are most likely the result of whole genome duplication events, the first of which probably occurred before the common ancestor of all extant angiosperms 62 , giving rise to C- and D-function genes with specialized functions 63 . Since Amborella is a basal angiosperm and contains only two genes in the AGAMOUS subfamily, one of each C- and D-function, it is highly probable that at least one copy of C- and D-function genes were present in the most basal angiosperm, which got expanded in higher eudicots probably because of gene and/or genome duplication events. The E-function of floral organ identity in Arabidopsis is carried out by AGL2 -like genes: AGL2, AGL3, AGL4, and AGL9 64–67 . The A. trichopoda genome has two AGL2- like genes: AmTrH2.10G036200 and AmTrH2.06G043000 , which we named AmtrAGL4 (AmtrSEP2) and AmtrAGL9 (AmtrSEP3) , respectively based on our orthology assessment. Two SEPALLATA (SEP) homologs were identified in Amborella and named AmtrAGL2 (SEP1) and AmtrAGL9 (SEP3) in a previous study 68 . An analysis of SEP homologs across a wide range of taxa showed that SEP1 and SEP2 homologs are restricted to Brassicaceae, while SEP4 is present only in core eudicots 69 . While our results also do not show the presence of SEP1 and SEP4 orthologs in Amborella , the presence of a close SEP2 ortholog does not align with the previous findings. Only one AGL2- like gene ( PRMADS1 ) was found in Pinus radiata 70 . However, since no other gymnosperm was found to have an AGL2 homolog, and PRMADS1 nested with an Eucalyptus AGL2- like gene in a phylogenetic tree, caution was advised when considering PRMADS1 as an AGL2 homolog 61 . Other basal angiosperms like Magnoliids and the basal eudicot Eschscholzia californica were also found to have two SEP homologs 68 . These observations suggest that SEP genes may have first originated in the common ancestor of all angiosperms. The SEP clade is considered to be sister to the AGL6 clade 61 , which contains AGL6 and AGL13 from Arabidopsis . AGL6 homologs are also found in gymnosperms 61 . Our analysis revealed one Amborella gene belonging to the AGL6 clade, and the AGL6 , SEP , and AP1/CAL/FUL clades formed a superclade. Sister clades AP1/FUL/SQUA and AGL6-SEP arose due to duplication events during evolution, of which only the AGL6 subfamily was retained in gymnosperms, while angiosperms retained the other subfamilies as well 71 . A. trichopoda has seven MIKC*-type genes, two of which share sequence homology with Arabidopsis MIKC* genes: 1) AmTrH2.04G150100 is now named AmtrAGL66/67/104 , and 2) AmTrH2.04G179300 is named AmtrAGL30 in the present study. Four MIKC* genes were first reported in the moss Physcomitrella patens , and they differ from MIKC C genes by the presence of a longer I-domain and have variable length and hydrophobic residues in the K-domain 12 . An elongation of the I-domain in the ancestral MIKC C -type gene was proposed to have given rise to the MIKC* lineage 12 , however later studies suggested that the MIKC* lineage was formed from a duplication in the region encoding the K-domain 72 . MIKC* genes have since been identified across all green plant lineages, from mosses to eudicots, and they are highly conserved in both structure and function from ferns to seed plants, although they are fewer in number in all plants compared to their MIKC C counterparts. It has been suggested that in most plant lineages, the MIKC* group contains two monophyletic clades, S and P, the origin of which can be traced back to more than 380 MYA to the ancestor of ferns and seed plants 73 . In the present study, the MIKC* subclade could be divided into two groups that had genes orthologous to Arabidopsis MIKC* genes, and a subclade that contained only A. trichopoda genes. Interestingly, only AmTrH2.08G064100.1 had a non-zero read count in mature flowers, without differential expression between the sexes, suggesting that these genes may have escaped identification. Unlike other MIKC*-genes, they do not appear to be expressed in the male gametophyte, or they could potentially be pseudogenes. The apparent lack of 5’ and 3’ untranslated regions (UTRs) in these genes (Fig. 4 ) supports the possibility that they could in-fact be pseudogenes. Also, the presence of two MIKC*-type genes: AmtrAGL30 and AmtrAGL66/67/104 in genomic blocks collinear with Arabidopsis (Fig. 7 ), suggests a strong functional conservation of these genes across plant lineages. Regarding Type I (Mα) MADS-Box genes, three paralogous genes on chromosome 1 ( AmTrH2.01G097100.1, AmTrH2.01G097000.1 : 1.07 and AmTrH2.01G096900.1, AmTrH2.01G097000.1 : 1.08) exhibited Ka/Ks values of > 1, indicating positive selection, but no Type II genes had the ratio > 1. Type-I MADS-Box genes are known to evolve faster than Type-II genes and experience faster birth and death rates 74 . Unlike Type-II genes, the functions of Type-I genes are still largely unexplored, and they are underrepresented in EST libraries across different plant species 14 . Despite some Arabidopsis Type-I genes being assigned with putative functions, most do not seem to have a functional restraint against non-synonymous mutations, which might explain them having a > 1 Ka/Ks value. Selection pressure in MADS-Box genes are generally known to favor purifying selection in other plant species as well 75–78 . Gene Structures and Motif Composition of A. trichopoda MADS-Box genes/proteins Our conserved motif analysis revealed that all MIKC C proteins in A. trichopoda have the typical MIKC domain pattern characteristic of Type-II MADS-Box genes. This included AmtrAGL12 , which is an Arabidopsis ortholog without coiled-coil structure because of lack of some hydrophobic residues 13 . The K-domain, unique to plant Type-II MADS-Box proteins, is not present in other eukaryotes, and likely evolved in the plant Type-II lineage after plant Type-II genes branched off from animal type-II genes 13 . Type-I Amborella MADS-Box genes have only one or two exons, while Type II genes have more complex structure with six to nine exons. This trend holds true across all plant MADS-Box genes, where Type I MADS-Box genes in plants have a much simpler gene structure with 1–2 exons than Type II, which typically have 6–8 exons 79 . The shorter length and simpler structure of Type I MADS-Box genes might have contributed to higher frequency of small-scale duplications than Type-II genes 80 . The diversity of Type II genes in higher plants is probably because of whole-genome duplication events, and retention of the duplicated genes for neofunctionalization, functional subdivision, or balancing the number of genes required for multimerization 74 . The fact that Type-I genes in Arabidopsis are characterized by 1 or 2 exons, and sometimes no intron, and their apparent lack of functionality suggests that Type-I genes are results of reverse transcription and most are pseudogenes without function 14 . Expression of MADS-Box genes in A. trichopoda floral buds and flowers Analysis of gene expression in floral buds and mature flowers of Amborella showed that many MIKC-type genes expressed differentially in males and females. This is in line with the current knowledge that floral transition and patterning genes belong to the Type-II group. The only Type-I genes with differential expression between the two sexes were AmTrH2.04G040100.1 , an Mg gene, which was upregulated in mature male flowers, and AmTrH2.09G063800.1 , an Ma gene, which was upregulated in female floral buds. Expression of Type-I MADS-box genes was not detected in any Arabidopsis tissue examined with microarrays and northern hybridization, which led researchers to conclude that Type-I genes could be non-functional in Arabidopsis 81 . A similar observation of low expression of Type-I genes was reported in another study, leading to an assumption that these genes either have very low expression levels or are expressed under very specific conditons 14 . The first type-I gene to be functionally characterized in Arabidopsis was AGL80 , also known as FEM111 , and it belongs to the Mg clade. agl80 mutants were found to affect female gametophytes after the fusion of polar nuclei with effects on nuclear maturation and vacuole size maintenance 82 . AGL80 was found to interact with the Ma protein AGL61, or DIANA (DIA), in maintaining female gametophyte development in Arabidopsis 83 . In Amborella , AmTrH2.09G063800.1.p did not phylogenetically nest in the clade with AGL61, but it was related to AmtrAGL62 (Fig. 1 ), and the gene was upregulated in female floral buds. There is no information in published literature about Ma genes and their expression in floral buds, and this could be a subject of further experimentation. However, several studies have linked Ma genes like AGL61/62 to endosperm, embryo, and female gametophyte development 84–87 . Expression analysis in floral buds and mature flowers also revealed some differentially expressed genes that were upregulated in different sexes at various stages. AmtrPI-2 and AmtrAP3-2 were upregulated in female buds and male flowers; and AmTrH2.11G126900.1 and AmtrAGL32 that were upregulated in male buds and female flowers. The first two are orthologs of B-function MADS-Box genes, responsible for petal and stamen identity in higher eudicots like Arabidopsis 5,8,55 . AmTrH2.11G126900.1.p nested in the AG/SHP/STK clade in our ML Tree (Fig. 1 ) and is potentially a C- or D-function ortholog. AmtrAGL32 , on the other hand, is a potential B-sister ortholog. Why B-function genes express differently in these different stages is difficult to explain with available data and literature, warranting further experimentation. Inrestingly, AmTrH2.11G126900.1 showed more than two-fold normalized expression difference between male and female floral buds (log2FC = 1.05), and much higher (~ 16-fold) upregulation in mature female flowers (log2FC = 4.09). This upregulation in female flowers could be explained if it were in fact a D-function ortholog specifying ovule identity, but explaining its upregulation in male floral buds remains unclear. Similarly. AmtrAGL32 showed near 21-fold upregulation in male floral buds (log2FC = 4.51) and much higher (~ 154 fold) upregulated in female flowers (log2FC = 7.26). In Arabidopsis , AGL32 , also known as ABS ( ARABIDOPSIS B SISTER ) or TRANSPARENT TESTA 16 ( TT16 ), is implicated in female-specific pathways such as proanthocyanidin biosynthesis in the seed coat 88 , cell patterning in the sub-epidermal integument cell layer 89 , in the nucellus cell death program 90 , co-ordination of cell division in ovule and seed coat and endosperm formation 91 . Upregulation of AmtrAGL32 in female flowers hints at similar roles of the ortholog in the basal angiosperm. Its upregulation in male floral buds remains unexplained well. AmtrAGL15 was downregulated in female buds too. In Arabidopsis , AGL15 and its paralog AGL18 are implicated in somatic embryogenesis 92–94 and flowering inhibition 95,96 . Hence, a higher transcript level of the gene in male floral buds could hint at distinct roles in the two sexes, a hypothesis supported by the fact that AmtrAGL15 was downregulated in female flowers. It could also be because of sampling errors if the male buds were collected at a slightly earlier developmental stage than the female buds. However, AGL18 known to be expressed in pollen 97 , could explain AmtrAGL15 ’s downregulation in female flowers. We found no differential expression of AmtrAGL20/42/71/72 between male and female floral buds. AGL20 , known as Suppressor of Overexpression of Constans 1 ( SOC1 ), is one of the earliest detected MADS-Box genes detected in the apical meristems of mustard after photoperiod-induced flowering 98 . The SOC1 mutants exhibit delayed flowering under both long- and short-day conditions 99 . SOC1 expression can also be induced by vernalization-induced inhibition of AGL25 or FLC ( FLOWERING LOCUS C ), a flowering repressor 100 , and acts as a common point for flowering signals from autonomous, vernalization, and photoperiod pathways 101 . We found no AGL25 ( FLC ) ortholog in Amborella , so it is possible that the floral transition pathway in the basal angiosperm could follow a different pattern than that seen in higher eudicots. This is especially interesting considering how floral organs are spirally arranged in Amborella , as opposed to whorled in higher eudicots. AmtrAGL22 , an ortholog of AGL22 or Short Vegetative Phase ( SVP ), also, showed no differential expression in floral buds of the two sexes, implying a conserved function of the gene in both sexes’ floral buds. AGL24 is another gene in the SVP clade in Arabidopsis , the ortholog of which was not present in Amborella . SVP , like AGL24 , regulates SOC1 expression too, but is a flowering repressor unlike AGL24 , despite being phylogenetically related 102 . The absence of an AGL24 ortholog further supports that the mechanism of floral transition in Amborella could be different from that in higher eudicots. AmtrAGL7/8/10/70 , the sole gene in the AP1/CAL/SQUA/FUL subfamily in Amborella , showed no differential expression between male and female floral tissues. AGL8 has a sequence very similar to AP1 ( AGL7 ) and CAL , and it has been found to act redundantly with AP1 and CAL to promote flower formation in Arabidopsis 103 . AmtrAGL7/8/10/70 did not have differential expression patterns between the two sexes, which hints at a function similar to that of FUL in Arabidopsis and its involvement in floral transition in both sexes with similar expression levels. In Arabidopsis, AGL8 expression is negatively regulated by AP1 expression during the formation of floral meristem and early stages of flower formation/floral organ identity, but AGL8 expression persists in the carpel walls and inflorescence meristems 104 . Since Amborella lacks distinct AP1 and AGL8 orthologs, it is likely that the regulation of A-function genes in Amborella differs from that in higher plants. Also, the absence of a distinct AP1 ortholog in Amborella allows us to make some assumptions about floral primordia formation in the species with spiral floral organs. In higher angiosperms, inflorescence meristems contain spirally arranged primordia, while floral meristem primordia are whorled, corresponding to whorled arrangement of the different floral axes 104 . In absence of more than one gene in the AGL7 family, primordia in the floral meristem might be established spirally, as with inflorescence meristem, hence producing flowers with spirally arranged floral organs. A study showing the presence of AP1 orthologs in basal angiosperms with spirally arranged stamens and carpels, such as Magnolia wufengensis 105 , conflicts with this hypothesis, however. The study cloned the AP1 ortholog in M. wufengensis by PCR from a cDNA library, and it was found that MawuAP1 could accelerate flowering and regulate carpel development in MawuAP1- expressing wild Arabidopsis but could not recover sepal and petal formation in Arabidopsis ap1 mutants. However, the study classified MawuAP1 as a FUL-like gene (supplementary data 105 ). Because MawuAP1 could not recover AP1 function in ap1 mutants, and because of high sequence similarity between AP1 and FUL genes, it is possible that the cloned MawuAP1 gene is more closely related to FUL than to AP1 , and this can explain spiral arrangement of floral organs in M. wufengensis as well. Based on these observations, we reaffirm our hypothesis that AP1 orthologs were probably absent in basal angiosperms with spirally arranged floral organs, and FUL-like genes represent the common ancestral state of the AP1/CAL/FUL subfamily in angiosperms. The differential expression of B-function ( AmtrAP3-1, AmtrAP3-2, AmtrPI-1, AmtrPI-2 ) orthologs in flowers of the two sexes is our notable finding. Both PI orthologs are upregulated in males, suggesting a conserved function of PI genes in the basal angiosperm in stamen identity similar to what is seen in higher eudicots. However, upregulation of AmtrPI-1 in female floral buds suggests that this PI ortholog might have some “rudimentary/atavistic” female expression as described by Becker et al. for some B-function genes 58 . This might also be the case with AmtrAP3-1 , where the gene retains an ancestral B-function of being expressed in females. The two genes encode nearly identical proteins but have different lengths because of intronic differences, as described previously. We looked at the cis -element differences between the two genes and found several elements that were unique to both of them, such as 3-AF1 binding site, ARE, AuxRR-core, LAMP-element, W-box, TCCC-element that were unique to AmtrAP3-1 and ABRE-4, AE-Box, as-1, TGA-element, MYB-like sequence unique to AmtrAP3-2 ’s promoter regions. The presence of some unique hormone-, light- and stress-responsive cis -elements in the two genes suggest environmental influences in sex-specific expression of AP3 paralogs in Amborella , warranting experimental verification. Upregulation of potential D-function ( AmTrH2.11G126900.1 ) and E-function ( AmtrAGL4 and AmtrAGL 9 ) upregulation in female flowers, and no differential expression of the C-function ortholog ( AmtrAG ) between the two sexes are indicative of Amborella plant employing a modification of the fading borders model of floral patterning in basal angiosperms. Kim et al.(2005), studied expression levels of the floral patterning orthologs in different floral organs of basal angiosperms 23 : the expression of “ Am.tr.PI” and “ Am.tr.AP3” was high in all floral organs (perianth, stamens, and carpels), and’the expression of “ Am.tr.AG “ was the highest in inner stamens and carpels, and Am.tr.AGL2 was expressed in all floral organs of Amborella , just prior to anthesis. Kim et al. (2004) also cloned two “ Am.tr.AP3” orthologs and reported the absence of the C-terminal domain in “ Am.tr.AP3-2” 55 . Our analysis revealed that both AP3 orthologs had complete MIKC domains, likely because we identified the genes using genome data while Kim et al used the transcriptome to characterize the genes. One AP3 ortholog present in the previous scaffold-level assembly of Amborella , however, lacks a C-terminus as well (data not shown). We find the combination of Kim et al.’s RT-PCR and our RNASeq-based approaches useful in enhancing our understanding of floral patterning in Amborella concerning B-, C-, D-, and E-function genes. Male and female Amborella flowers show distinct expression patterns of B- and E-function genes, specifically AmtrAP3-2, AmtrPI-1 and AmtrPI-2 are upregulated in male flowers, while AmtrAP3-2 , AmtrAGL4 and AmtrAGL9 are upregulated in female flowers. The genetic consequences of these distinct expression patterns, and if these are associated with sex determination in Amborella , remain to be investigated. The C-function ortholog AmtrAG is expressed in the inner reproductive organs in the floral spiral in both sexes, while the D-function ortholog’s upregulation in female flowers, suggesting its function in ovule development, as in the classical ABCDE model. Why E-function orthologs are upregulated in female flowers is another question that can be researched further. Based on these observations, we propose that the Fading Borders model of floral patterning in Amborella employs different B-function orthologs in the two sexes, E-function orthologs are upregulated in females, while C- and D- function orthologs have functions similar to that in the ABCDE model observed in higher eudicots. Recent research by Carey et al. identified chromosome-9 as the location of sex-determining region (SDR) in the Amborella 106 . Interestingly, several MADS-Box genes that showed sex-specific up- or down-regulation in Amborella were found to be located on chromosome-9 in our study as well. Male-biased genes such as AmtrAP3-2 , AmtrAGL20/42/71/72 , and AmreAGL6/13 and female-biased genes AmtrAGL32 and AmTrH2.09G064500.1 were located on chromosome-9, suggesting a strong correlation of expression of MADS-Box genes with sex-determination in Amborella . The upregulation of the two MIKC*-type genes in Amborella ( AmtrAGL66/67/104 ) and ( AmtrAGL30 ) in male flowers hints at the conserved function of MIKC*-type MADS-Box genes in male gametophyte development. Except AGL67 which is expressed during late embryonic development, the other five MIKC* orthologs in Arabidopsis were found to express exclusively in the pollen, predominantly from the tricellular stage onward, which occurs after the second mitosis 107 . To summarize, we identified 42 MADS-Box genes in the Amborella trichopoda genome, classified and named them with reference to Arabidopsis thaliana orthologs. We could assign sequence-based orthology to 20 of these genes to Arabidopsis MADS-Box genes with Maximum-Likelihood and Reciprocal Best Hit BLAST methods and named them after the assigned orthologies. We conducted structural and functional analyses of the identified genes and based on expression data of floral buds and flowers, we found Type-II MADS-Box genes to be highly expressed, with several genes being differentially expressed between the two sexes, in the bud and mature flower stages, with floral transition-related genes’ and B- and E-function orthologs’ expression being highly dependent on the sex of the plant. Our results provide crucial data on updating the fading borders model of floral patterning in basal angiosperms with sex-specific gene expression patterns. Methods Taxonomy and Phylogeny of Amborella trichopoda MADS-Box Genes We retrieved the Amborella trichopoda genome ( Amborella trichopoda var. SantaCruz_75 HAP1 v2.1), coding sequences (CDS), annotation files, and proteome from Phytozome 108 and Arabidopsis thaliana MADS-Box sequences from The Arabidopsis Information Resource (TAIR) 46 . MADS-Box sequences in the A. trichopoda proteome were retrieved with two rounds of HMMER search with HMMER version 3.3.2 42 . For the first HMMER search, A. thaliana MADS-Box protein sequences were aligned using Muscle 5.1.linux64 109 accessed from Bioconda 110 , and used the aligned sequences to create a Hidden Markov Model (HMM) profile. This profile was used as the query against the A. trichopoda proteome with an expect threshold (E) value of 0.1. For the second HMMER search, the MADS-Box HMM profile (PF00319) was downloaded from the EBI-Interpro 111 database, which was used as the query against the A. trichopoda proteome in a HMMER search with an expect threshold (E) value of 0.1. The sequences were retrieved from the proteome fasta file with a custom-written bash script. We filtered the results from both searches for unique hits, and analyzed the sequences for the presence of the MADS- and K-signature domains against Pfam 45 , Simple Modular Architecture Research Tool (SMART) 44 , and National Center for Biotechnology Information (NCBI) Conserved Domain Database (CDD) 43 databases with InterProScan version 5.52-86.0 111 . For the phylogenetic analysis, we aligned the full-length MADS-Box protein sequences of A. trichopoda and A. thaliana using Muscle, with Chara globuralis MADS Box1 ( CgMADS1 ) 17 as the outgroup. Phylogenetic analysis was performed using Maximum Likelihood (ML) method in IQTREE2 112 with 1000 bootstraps with the best substitution model. The model (-m) option was set to TEST to choose the best substitution model for tree construction via ModelFinder 113 . The resulting tree file was visualized and annotated in Interactive Tree of Life (iTOL) version 6 114 . Homologies among A. trichopoda and A. thaliana MADS-Box proteins were inferred based on nesting of sequences with > 50 bootstrap support. For A. trichopoda scaffolds not resolved with the phylogenetic tree, we used a modification of the reciprocal best hit (RBH) method as employed by Bai et al. 78 . For the RBH method, a protein-protein blast (blastp) was carried out using full length sequences of all A. trichopoda MADS-box hits as queries against A. thaliana MADS-Box protein sequences with BLAST 115 . Another blastp was carried out with A. thaliana MADS-Box protein sequences as query against the A. trichopoda sequences as the database. Pairs with the best bitscores and E-values from the two BLAST runs were treated as putative orthologs. A. trichopoda MADS-Box genes were named based on their homologies with A. trichopoda MADS-Box genes, for instance, AmtrAGLx for a gene homologous to AGLx . A. trichopoda sequences that could not be assigned with orthologous A. thaliana sequences with both approaches were designated as not orthologous to any A. thaliana MADS-Box genes and were left unnamed. Analysis of Physicochemical Properties and Subcellular Localization We used the online ExPASy 116 tool to calculate protein lengths, molecular weights, and isoelectric points of A. trichopoda MADS-Box proteins. Bologna Unified Subcellular Component Annotator (BUSCA) 117 was used to predict subcellular localization of the proteins. Analysis of Conserved Motifs and Gene Structure Sequences filtered from the InterProScan search and CDD alignment were subjected to a conserved motifs analysis with Multiple Expectation maximizations for Motif Elicitation (MEME) version 5.4.1 118 with parameters: total number of motifs = 10, minimum motif width = 6 and maximum motif width = 100. We used the subset of the Gene Feature Format (gff3) file of the A. trichopoda genome to construct the exon-intron map of the A. trichopoda MADS-Box genes in TBtools-II 119 , accounting for the orientation of the genes. The constructed map was visualized and edited in Inkscape 1.3.2 ( https://inkscape.org/ ). Analysis of cis -regulatory Elements The 2000 basepair (bp) upstream regions of the identified MADS-Box genes were extracted from the genome fasta file utilizing the coordinates from the gff3 file with BEDTools 120 , accounting for orientation of the genes. A fasta file prepared with the 2000bp regions was used to identify cis -elements with PlantCARE 121 . The resulting file was analyzed, and the top 20 most common cis -elements were visualized in the promoter regions with TBTools-II. Gene Ontology Analysis The fasta files of the identified MADS-Box proteins was subjected to gene ontology analysis with BLAST2GO 122 . The results were visualized with the graphing functionality of MS-Excel. Chromosomal Locations, Collinearity and Evolutionary Selection Pressure Analysis Chromosomal locations of the MADS-Box genes were visualized with a subset of the genomic gff3 file with TBtools-II. Collinearity between the Amborella and Arabidopsis genomes was examined with the Multiple Collinearity Scan toolkit (MCScanX) 123 with whole-proteomes’ reciprocal BLASTP results, defining block size as 5, and the resulting collinearity file was used to generate a dual plot with TBTools-II showing MADS-Box genes in collinear blocks. Ka/Ks ratios among all possible gene combinations were calculated using the Simple KaKs calculator built in TBtools-II. Gene Expression Data Acquisition and Analysis RNASeq fastq files from BioProjects PRJNA748676 (floral buds) and PRJEB38698 (mature flowers) were retrieved from the European Nucleotide Archive (ENA) database 124 . The fastq files were quality-checked with FASTQC 125 and MULTIQC 126 , and trimmed with Btrim 127 as needed. The quality-controlled RNASeq fastq files were aligned against the index file created with A. trichopoda cds and genome fasta files, and a .txt decoy file created with scaffold ids of the genome with Salmon 128 to create transcript quantification files. The quantification (.sf) files generated from experiments coming from all biological replicates corresponding to a treatment were merged with the quantmerge function in Salmon with the --column argument assigned for numreads and tpm to generate two merged .sf files, which were converted to .csv files. Differential expression analyses were carried out with the DESeq2 package 129 from Bioconductor 130 in RStudio version 2024.4.1.748 131,132 . Expression data for MADS-Box genes from the normalized gene expression data were extracted with a custom R script. log2FC change and p-adjusted (padj) values of genes were plotted in volcano plots with the R package EnhancedVolcano 133 . Differentially expressed genes between treatments were identified based on log2FC values and adjusted p-values (padj). Expression heatmaps were generated using Z-scores of normalized transcripts per million (TPM) values from the raw read counts of MADS-Box genes, and visualized using the pheatmap 134 package from Bioconductor in RStudio. Principal Component Analysis was carried out in RStudio with base R commands, and visualized with the ggplot2 R package 135 . Declarations Conflict of interest The authors declare no conflict of interest. Funding This project is supported by the USDA-AFRI (Award # 2022-67037-36254) and South Dakota Agriculture Experiment Station Hatch Project #SD00H800-23 to M.P. Nepal Author Contribution S.P. wrote the codes and scripts, performed the analyses, and wrote the original manuscript. B.A. assisted in writing and reviewing the draft. M.P.N. conceived and supervised the project, frameworked the experiment and analyses, assisted writing of the original manuscript, reviewed, and finalized the manuscript. Acknowledgment Computational resources were provided by the High-Performance Computing (HPC) Cluster at South Dakota State University. Data Availability The protein and DNA sequences analyzed in this study are accessible in the Amborella trichopoda genome in Phytozome (Phytozome genome ID: 727) (https://phytozome-next.jgi.doe.gov/info/Atrichopodavar_SantaCruz_75HAP1_v2_1). 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Supplementary Files SupplementaryFileS2.xlsx SupplementaryFileS3.xlsx SupplementaryFileS4.xlsx SupplementaryFileS5.xlsx SupplementaryFileS6.xlsx SupplementaryInformation.docx Cite Share Download PDF Status: Published Journal Publication published 12 Feb, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 12 Jan, 2025 Reviews received at journal 26 Nov, 2024 Reviewers agreed at journal 17 Nov, 2024 Reviewers agreed at journal 14 Nov, 2024 Reviewers invited by journal 12 Nov, 2024 Editor assigned by journal 12 Nov, 2024 Editor invited by journal 11 Nov, 2024 Submission checks completed at journal 11 Nov, 2024 First submitted to journal 22 Oct, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5314709","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":382854577,"identity":"f1c37913-0e39-44b1-a356-b861bc769d1c","order_by":0,"name":"Sanam Parajuli","email":"","orcid":"","institution":"South Dakota State University","correspondingAuthor":false,"prefix":"","firstName":"Sanam","middleName":"","lastName":"Parajuli","suffix":""},{"id":382854578,"identity":"79240017-15d2-401b-a06e-b39242d46504","order_by":1,"name":"Bibek Adhikari","email":"","orcid":"","institution":"South Dakota State University","correspondingAuthor":false,"prefix":"","firstName":"Bibek","middleName":"","lastName":"Adhikari","suffix":""},{"id":382854579,"identity":"eb2ff7f2-09b2-4bfc-9e9b-ef98e83d1497","order_by":2,"name":"Madhav P. Nepal","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBElEQVRIiWNgGAWjYHACMxiD8cAHBmYYhxm7YjQtDAdnAFXyQFQTqeUwDzFadNsPb3vws40hj1+6+cFhmz/W9vbs5w8+YKiwTmzAZcWZtHLD3jaGYsk5xwwO57alJ/bwJDMbMJxJx63lQI6ZBM8ZhsQNNxKAWhoOJ/AwJLNJMLYdxq3l/BszyT9ALftvpH84bPHnsD0P/2P2H4z/8Gi5kWMmzVMBtEUix+AwA9thxh6JZDYGxgZ8Wp6VSctUSCTOuHOm4GAvyC83HhtLJBxLN8btsORtkm8MbBL7Z7dvfPADGGLs/YkPP3yosZbFpQUKJMAIARLwK0foGgWjYBSMglGAFQAAN7NcRpBXKMQAAAAASUVORK5CYII=","orcid":"","institution":"South Dakota State University","correspondingAuthor":true,"prefix":"","firstName":"Madhav","middleName":"P.","lastName":"Nepal","suffix":""}],"badges":[],"createdAt":"2024-10-23 00:53:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5314709/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5314709/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-88880-x","type":"published","date":"2025-02-12T15:57:35+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":70097996,"identity":"73025f34-7e41-4bde-bc31-f3884797600d","added_by":"auto","created_at":"2024-11-28 10:02:21","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1773936,"visible":true,"origin":"","legend":"\u003cp\u003eMaximum Likelihood tree depicting the relationships among 42 identified Amborella trichopoda and 105 Arabidopsis thaliana MADS-Box proteins. The tree was constructed with 1000 bootstrap replications, with Chara globuralis MADS-Box protein (CgMADS1) as the outgroup. The best-fit model for tree was identified as JTT+F+G4. The numbers at the nodes indicate bootstrap values in percentage. Amborella trichopoda proteins are displayed in bold. The classification and nomenclature of A. trichopoda proteins were determined with reference to Arabidopsis MADS-Box proteins, based on bootstrap support (\u0026gt;50) in the tree and the Reciprocal Best Hit (RBH) analysis described in the Methods section. A. trichopoda proteins with unresolved orthologies were not assigned a name. Different colors refer to the different classes as shown in the legend alongside the figure.\u003c/p\u003e","description":"","filename":"floatimage178.png","url":"https://assets-eu.researchsquare.com/files/rs-5314709/v1/6c6f6d1ff6b4a423ade04126.png"},{"id":70098865,"identity":"f645fc69-4143-4d2c-9017-15b63eb774b9","added_by":"auto","created_at":"2024-11-28 10:10:21","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":340994,"visible":true,"origin":"","legend":"\u003cp\u003eConserved motif composition of A. trichopoda MADS-Box proteins. Proteins are listed following their classification in the left column and the motif composition is visualized with boxes of different colors. The protein sequences are oriented from the N-terminus on the left to the C-terminus on the right.\u003c/p\u003e","description":"","filename":"floatimage259.png","url":"https://assets-eu.researchsquare.com/files/rs-5314709/v1/363992ef84171d584c4ff172.png"},{"id":70100577,"identity":"86378d65-43d5-4a7d-a1b3-0c39ead3e043","added_by":"auto","created_at":"2024-11-28 10:26:22","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":241383,"visible":true,"origin":"","legend":"\u003cp\u003eCoding Sequences (CDS) and Untranslated Regions (UTRs) composition of MADS-Box genes in Amborella trichopoda. Genes run from the 5’ to 3’ direction (left to right), with measurements in base pairs (bp), and are placed in the left column following their classification. Structural elements are displayed with different colored rectangular boxes as indicated in the legend panel. Introns are represented by lines connecting the CDS boxes.\u003c/p\u003e","description":"","filename":"floatimage353.png","url":"https://assets-eu.researchsquare.com/files/rs-5314709/v1/2513d91561b1a4da71db2803.png"},{"id":70097431,"identity":"e8deb6cc-3cac-4a8d-bea4-562c1794c560","added_by":"auto","created_at":"2024-11-28 09:54:22","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":485764,"visible":true,"origin":"","legend":"\u003cp\u003eCo-ordinates of the top 20 cis-elements in the 2000bp upstream regions of Amborella trichopoda MADS-Box genes, visualized by occurrence frequency. Different cis-elements are color-coded as shown in the legend panel on the right. The X-axis represents the gene sequences running from the 5’ to 3’ direction.\u003c/p\u003e","description":"","filename":"floatimage439.png","url":"https://assets-eu.researchsquare.com/files/rs-5314709/v1/2b66d678a97f14acf061e9aa.png"},{"id":70097425,"identity":"a3e7009a-96d5-4cdd-a8f0-fba32ffb56ee","added_by":"auto","created_at":"2024-11-28 09:54:21","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":62506,"visible":true,"origin":"","legend":"\u003cp\u003eGene Ontology (GO) annotations assigned to the identified Amborella trichopoda MADS-Box proteins. Labels on the X-axis are the Assigned GO terms are on the X-axis while the number of genes associated with each ontology is on the Y-axis.\u003c/p\u003e","description":"","filename":"floatimage511.png","url":"https://assets-eu.researchsquare.com/files/rs-5314709/v1/a8b85b3526224e4299efb825.png"},{"id":70097427,"identity":"c45f18c7-c8e9-4d2b-8dde-eb054956e2b3","added_by":"auto","created_at":"2024-11-28 09:54:21","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":205139,"visible":true,"origin":"","legend":"\u003cp\u003eChromosomal locations of the 42 Amborella trichopoda MADS-Box genes. Chromosomes are labeled on the right side of their respective representations, with genes labeled in red on the left. The scale on the leftmost end shows chromosome lengths in mega-base pairs (Mb).\u003c/p\u003e","description":"","filename":"floatimage620.png","url":"https://assets-eu.researchsquare.com/files/rs-5314709/v1/d9ee70923560af20aa4c2072.png"},{"id":70099215,"identity":"a4870089-51e9-49fa-9bcd-db666da72450","added_by":"auto","created_at":"2024-11-28 10:18:22","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":516849,"visible":true,"origin":"","legend":"\u003cp\u003eDual synteny plot showing all collinear blocks between Amborella trichopoda (blue, am) and Arabidopsis thaliana (green, at) chromosomes, with gray connectors. Block-connectors with collinear MADS-Box genes are highlighted in red.\u003c/p\u003e","description":"","filename":"floatimage711.png","url":"https://assets-eu.researchsquare.com/files/rs-5314709/v1/824d218022cae1869a3bff8a.png"},{"id":70098001,"identity":"eaf95045-74dc-4159-8956-5cf66fd1a519","added_by":"auto","created_at":"2024-11-28 10:02:22","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":160625,"visible":true,"origin":"","legend":"\u003cp\u003eVolcano plot showing differentially expressed genes in Amborella female flowers with reference to male flowers. The X-axis represents log2 fold change (log2FC) gene expression values, a higher log2FC value meaning a higher fold-change. The Y-axis represents the respective -log10 adjusted p-adjusted values, higher values meaning higher statistical significance. Only genes with padj values of \u0026lt; 0.05 and log2FC values of \u0026gt;1 have been labeled. Genes upregulated in female flowers are plotted on the right while those downregulated have been plotted on the left.\u003c/p\u003e","description":"","filename":"floatimage84.png","url":"https://assets-eu.researchsquare.com/files/rs-5314709/v1/bb0920e5008a9b80fd773a06.png"},{"id":70097442,"identity":"2b3b8421-9c1a-4717-96c9-27078b9c999e","added_by":"auto","created_at":"2024-11-28 09:54:22","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":114308,"visible":true,"origin":"","legend":"\u003cp\u003ePrincipal Component Analysis (PCA) plot showing the separation of male and female samples based on expression profiles of MADS-Box genes in mature male and female flowers. Each point represents an individual sample, with samples labelled F for female and M for male. PC1 explains 50.12% of the observed variance while PC2 explains 10.37% of the observed variance. Samples and 95% Confidence Interval (CI) ellipses are color-coded by sex, as shown in the legend alongside. Samples for each sex cluster tightly, indicating consistent expression of MADS-Box genes within sexes.\u003c/p\u003e","description":"","filename":"floatimage94.png","url":"https://assets-eu.researchsquare.com/files/rs-5314709/v1/7a387801c37dfea7076bcc5b.png"},{"id":70097440,"identity":"d4a3d72e-5806-4812-ab7a-c7fac3888d9f","added_by":"auto","created_at":"2024-11-28 09:54:22","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":18874,"visible":true,"origin":"","legend":"\u003cp\u003eHeatmap of z-transformed Transcripts Per Million (TPM) values of MADS-Box genes expressed in male and female mature flower samples. The horizontal axis shows the different sample names while the vertical axis are the different MADS-Box genes. Clustering of genes and samples is based on Euclidean distance calculated from the expression patterns.\u003c/p\u003e","description":"","filename":"floatimage1011.png","url":"https://assets-eu.researchsquare.com/files/rs-5314709/v1/b15882097127094ebeab648d.png"},{"id":70098871,"identity":"0dde528c-a5b9-49f8-965c-4d6c0b5eae5c","added_by":"auto","created_at":"2024-11-28 10:10:22","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":104584,"visible":true,"origin":"","legend":"\u003cp\u003eVolcano plot showing differentially expressed genes in female Amborella floral buds with reference to male floral buds. The X-axis represents log2 fold change (log2FC) values with higher l values meaning greater fold-change. The Y-axis represents the respective -log10 adjusted p- values, higher values meaning greater statistical significance. Only genes with padj values of \u0026lt; 0.05 and log2FC \u0026gt;1 are labeled. Genes upregulated in female floral buds are plotted on the right while those downregulated are plotted on the left.\u003c/p\u003e","description":"","filename":"floatimage112.png","url":"https://assets-eu.researchsquare.com/files/rs-5314709/v1/08d87785c8b93986fe22a0a8.png"},{"id":70097443,"identity":"57659ece-84c8-4545-881b-187ad4fac998","added_by":"auto","created_at":"2024-11-28 09:54:22","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":153520,"visible":true,"origin":"","legend":"\u003cp\u003ePrincipal Component Analysis (PCA) plot showing the separation of male and female samples based on expression profiles of MADS-Box genes in male and female floral buds. Each point represents an individual sample, with samples labelled F for female and M for male. PC1 explains 50.12% of the observed variance whole PC2 explains 10.37% of the observed variance. Samples and 95% Confidence Interval (CI) ellipses are color-coded by sex, as shown in the legend alongside. An overlap in the 95% CIs between the sexes hints at a weaker distinction between sexes in terms of the expression pattern of MADS-Box genes in floral buds.\u003c/p\u003e","description":"","filename":"floatimage121.png","url":"https://assets-eu.researchsquare.com/files/rs-5314709/v1/7fa8542e71d23dd630b916c9.png"},{"id":70099216,"identity":"be943432-0318-461a-8e97-32ebf6aaf93c","added_by":"auto","created_at":"2024-11-28 10:18:22","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":19620,"visible":true,"origin":"","legend":"\u003cp\u003eHeatmap of z-transformed Transcripts Per Million (TPM) values of MADS-Box genes expressed in male and female floral bud samples. Sample names are on X-axis while MADS-Box genes are on Y-axis. Clustering of genes and samples is based on Euclidean distance calculated from the gene expression patterns.\u003c/p\u003e","description":"","filename":"Onlinefloatimage13.png","url":"https://assets-eu.researchsquare.com/files/rs-5314709/v1/4897ce1fbd59175e609de46c.png"},{"id":76487561,"identity":"d6f617a7-aa7b-43df-a87f-97b0cfa4891c","added_by":"auto","created_at":"2025-02-17 16:09:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5431406,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5314709/v1/5672c4b6-1920-4c29-97ca-5efd65079536.pdf"},{"id":70097997,"identity":"37ec6dc0-b029-4b02-8462-096ae1093d0a","added_by":"auto","created_at":"2024-11-28 10:02:21","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":580250,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFileS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5314709/v1/293ff798e8d7e32a835edcce.xlsx"},{"id":70098868,"identity":"3f1382da-5bf6-42cf-8819-1d03479b6d99","added_by":"auto","created_at":"2024-11-28 10:10:22","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":17309,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFileS3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5314709/v1/1c3f1844432dda01994a50f2.xlsx"},{"id":70098005,"identity":"8c4194c0-5b9a-4510-9282-673af9177a41","added_by":"auto","created_at":"2024-11-28 10:02:22","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":265927,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFileS4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5314709/v1/fc2a7dd54b4e481b9735e858.xlsx"},{"id":70098004,"identity":"dda5dee4-bdc0-4d28-9ecf-49390a08669a","added_by":"auto","created_at":"2024-11-28 10:02:22","extension":"xlsx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":106115,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFileS5.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5314709/v1/ddd62e89b20ad7d53a012513.xlsx"},{"id":70098008,"identity":"e993590d-2c9a-4e09-93bb-43373b649e20","added_by":"auto","created_at":"2024-11-28 10:02:22","extension":"xlsx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":18547067,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFileS6.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5314709/v1/f101d66f5afc1079fe68ba59.xlsx"},{"id":70097441,"identity":"a86f8ab1-fd5a-44a4-a0f7-c50b75ef1a8d","added_by":"auto","created_at":"2024-11-28 09:54:22","extension":"docx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":15850,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-5314709/v1/c29df8aba9495dc62451a389.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Insights into Genetics of Floral Development in Amborella trichopoda Baill through Genome-wide Survey and Expression Analysis of MADS-Box Transcription Factors","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePlants undergo a vegetative to reproductive switch in their life cycle, and the onset of this switch is influenced by environmental cues, along with several endogenous developmental factors\u003csup\u003e1\u003c/sup\u003e. These signals are carefully timed for resources like pollinator activity, water availability, wind, etc. crucial for the plants\u0026rsquo; reproductive success\u003csup\u003e2\u003c/sup\u003e. This transition replaces vegetative meristems with floral meristems, after which cells in the floral meristem differentiate into tissues that make up the floral whorls: calyx, corolla, androecium, and gynoecium in a perfect flower. The genes regulating this patterning were first identified in \u003cem\u003eAntirrhinum majus\u003c/em\u003e\u003csup\u003e3\u003c/sup\u003e and \u003cem\u003eArabidopsis thaliana\u003c/em\u003e\u003csup\u003e4\u003c/sup\u003e with homeotic mutants, where one floral organ is replaced by another as a result of mutation(s) in key regulatory genes. With systematic analyses of these mutations, the ABC model of floral patterning was proposed in 1991\u003csup\u003e5\u003c/sup\u003e. This model explains that three classes of genes contribute to floral patterning with overlapping whorls of expression: sepal identity is determined by A-function genes (e.g., \u003cem\u003eArabidopsis APETALA1, AP1\u003c/em\u003e and \u003cem\u003eAP2\u003c/em\u003e), petals by A- and B- function genes (e.g., \u003cem\u003eArabidopsis AP3\u003c/em\u003e and \u003cem\u003ePISTILLATA\u003c/em\u003e, \u003cem\u003ePI\u003c/em\u003e), androecium by B- and C- function genes (e.g., \u003cem\u003eArabidopsis AGAMOUS\u003c/em\u003e, \u003cem\u003eAG\u003c/em\u003e), and gynoecium by C-function genes\u003csup\u003e5\u003c/sup\u003e. Later, the model was expanded to the ABCDE model after the discovery of D-function genes (e.g.: \u003cem\u003eArabidopsis AGAMOUS-LIKE11\u003c/em\u003e, \u003cem\u003eAGL11\u003c/em\u003e) that determine ovule identity\u003csup\u003e6\u003c/sup\u003e and E-function genes (e.g., \u003cem\u003eArabidopsis SEPALLATA1, SEP1\u003c/em\u003e to \u003cem\u003eSEP4\u003c/em\u003e) that acted in conjunction with B- and C-function genes in their respective whorls\u003csup\u003e7\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAll floral identity and patterning genes, except \u003cem\u003eA. thaliana AP2\u003c/em\u003e and its orthologs, have a highly conserved domain known as the MADS-Domain and are collectively called floral MADS-Box genes\u003csup\u003e8\u003c/sup\u003e. The abbreviation MADS refers to a group of genes from different organisms: \u003cem\u003eMINICHROMOSOME MAINTENANCE1\u003c/em\u003e (\u003cem\u003eMCM1\u003c/em\u003e) from yeast, \u003cem\u003eAGAMOUS\u003c/em\u003e (\u003cem\u003eAG\u003c/em\u003e) from \u003cem\u003eArabidopsis\u003c/em\u003e, \u003cem\u003eDEFICIENS\u003c/em\u003e (\u003cem\u003eDEF\u003c/em\u003e) from \u003cem\u003eAntirrhinum\u003c/em\u003e and Serum Response Factor (\u003cem\u003eSRF\u003c/em\u003e) from humans, all encoding for proteins with an approximately 60 amino acid-long DNA-binding MADS-Domain in eukaryotes\u003csup\u003e9\u003c/sup\u003e. Genes containing the MADS or the M-domain collectively form the MADS-Box gene family. Proteins encoded by these genes are transcription factors that bind to promoter regions with a highly conserved nucleotide motif called the CArG-box (C-A-rich-G-box) with the consensus sequence 5\u0026rsquo;-CC(A/T)\u003csub\u003e6\u003c/sub\u003eGG-3\u0026rsquo;, or other similar sites like the \u0026ldquo;N-10 type CArG box\u0026rdquo; or the Monocyte Enhancer Factor2 (MEF2) consensus binding site\u003csup\u003e10\u003c/sup\u003e. The dependence of floral identity on MADS-Box homeotic genes clearly puts duplication and diversification of these genes at the center of flower evolution\u003csup\u003e11\u003c/sup\u003e. Based on structural differences, MADS-Box proteins are broadly divided into two types: Type I and Type II. Type I genes are also called the M-type genes and Type II are known as MIKC-type, owing to their structure that contains the MADS (M), Intervening (I), Keratin-like (K), and C-terminal (C) domains\u003csup\u003e12\u003c/sup\u003e. Most Type I genes have a single exon and lack the K-box\u003csup\u003e10,13,14\u003c/sup\u003e. The K-box is an approximately 70 amino acid-long domain unique to plant\u0026rsquo;s MIKC-type MADS-Box proteins\u003csup\u003e10\u003c/sup\u003e and is believed to have evolved in the extant Streptophytes after the divergence from the common ancestor of plants and animals 700\u0026nbsp;million years ago (MYA)\u003csup\u003e15\u003c/sup\u003e. The Type-I genes are further classified as Ma, Mb, and Mg based on their phylogenetic nesting\u003csup\u003e16\u003c/sup\u003e, while Type-II genes are divided into MIKC\u003csup\u003eC\u003c/sup\u003e and MIKC*-types based on intron-exon structures, a longer I-domain, and a K-domain encoded by more exons in the MIKC*-type\u003csup\u003e12\u003c/sup\u003e, also known as Mδ genes\u003csup\u003e16\u003c/sup\u003e. Represented by a single MIKC-type gene in the Charophycean alga \u003cem\u003eChara globularis\u003c/em\u003e\u003csup\u003e17\u003c/sup\u003e, MADS-Box genes have greatly expanded in higher plants.\u003c/p\u003e \u003cp\u003e \u003cem\u003eAmborella trichopoda\u003c/em\u003e Baill., the sole member of the family Amborellaceae, is considered the sister species to all extant angiosperms, and hence has caught the attention of botanists and evolutionary biologists worldwide. Native to New Caledonia, this woody evergreen shrub has been the focus of numerous prominent evolutionary studies\u003csup\u003e18\u0026ndash;20\u003c/sup\u003e. \u003cem\u003eAmborella\u003c/em\u003e is a dioecious species. meaning individual plants bear either male or female flowers arranged in botryoids panicles that are poorly branched. Male flowers measuring 4-5mm are slightly larger than female flowers measuring 3-4mm in diameter, and both are creamy white in color, with all floral organs spirally arranged. Each flower is surrounded by two prophylls, followed by spirally arranged tepals (9\u0026ndash;11 in males, 7\u0026ndash;8 in females), forming the perianth. The number of stamens in the male flower can range from 12\u0026ndash;21, some inner ones in the spiral occasionally being sterile. Female flowers typically have five carpels and usually contain one or two staminodes, or sterile stamens, which still possess pollen sacs. However, pollen is not formed in female flowers as the pollen development ceases just before meiosis. Male flowers may also have an undifferentiated bulge in the center of the flower that is sometimes described as a rudimentary carpel\u003csup\u003e21\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eBecause the ABCDE model was based on observations on perfect flowers of higher angiosperms like \u003cem\u003eAntirrhinum\u003c/em\u003e, \u003cem\u003eArabidopsis\u003c/em\u003e, and \u003cem\u003ePetunia\u003c/em\u003e, it does not fully explain floral patterning in plants with simpler and unisexual flowers. In the case of \u003cem\u003eAmborella trichopoda\u003c/em\u003e, flower organs are spirally arranged, transitioning from bracts to tepals and stamens or carpels. The \u0026ldquo;Fading Borders\u0026rdquo; model of floral patterning was proposed in 2004 for \u003cem\u003eAmborella\u003c/em\u003e and other basal angiosperms, suggesting that floral organ identity genes are broadly expressed in the floral meristem but weakly expressed in the outer and inner edges\u003csup\u003e22\u003c/sup\u003e. Subsequent analyses revealed that \u003cem\u003eAP1\u003c/em\u003e (A-function) homologs are expressed in all floral organs and leaves, \u003cem\u003eAP3/PI\u003c/em\u003e (B-function) homologs are expressed in all floral organs, and \u003cem\u003eAG\u003c/em\u003e (C-function) homologs of basal angiosperms follow the classical ABC model in being expressed in both stamens and carpels\u003csup\u003e23\u003c/sup\u003e. However, the fading borders model does not explain the mechanism in dioecious plants like \u003cem\u003eAmborella\u003c/em\u003e, nor how the floral patterning genes function in unisexual flowers. The model was suggested based on expression data, which might not capture genes that are unexpressed or get expressed at undetectable levels. A scaffold-level \u003cem\u003eA. trichopoda\u003c/em\u003e genome was released in 2013 and 36 MADS-Box genes were predicted\u003csup\u003e24\u003c/sup\u003e. However, the identified genes were not characterized in detail for structure and expression, although protein-protein interactions among them were analyzed with hybrid assays. A recent study identified several \u003cem\u003eAmborella\u003c/em\u003e genes associated with male gametophyte development, which included MIKC-type MADS-Box genes\u003csup\u003e25\u003c/sup\u003e. MIKC\u003csup\u003eC\u003c/sup\u003e-type genes in \u003cem\u003eAmborella\u003c/em\u003e were identified in a more recent study, but identification of Type-I genes was outside its scope, and the identified genes were not characterized in detail\u003csup\u003e26\u003c/sup\u003e. A thorough genome-wide analysis and characterization study for MADS-Box genes in \u003cem\u003eA. trichopoda\u003c/em\u003e has not been conducted yet, despite the gene family\u0026rsquo;s immense importance in plant development functions. With the availability of a chromosome-level genome assembly, we now have an opportunity to explore the functional and evolutionary dynamics of these genes in this basal angiosperm. The primary objectives of this study were to conduct genome-wide identification and characterization of \u003cem\u003eA. trichopoda\u003c/em\u003e MADS-Box genes, establish their orthologous relationships with \u003cem\u003eA. thaliana\u003c/em\u003e MADS-Box genes, and analyze their expression in floral buds and mature flowers. This will help us elucidate the genetic mechanisms underlying floral transitions, floral patterning, and sex expression in \u003cem\u003eA. trichopoda\u003c/em\u003e.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eTaxonomy and Phylogeny of\u003c/b\u003e \u003cb\u003eA. trichopoda\u003c/b\u003e \u003cb\u003eMADS-Box Genes\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe identified 42 MADS-Box containing genes in the \u003cem\u003eA. trichopoda\u003c/em\u003e genome. Based on their phylogenetic relationships with \u003cem\u003eA. thaliana\u003c/em\u003e MADS-Box genes, they were classified into all the major identified groups of MADS-Box genes: 20 gene members belonged to the MIKC\u003csup\u003eC\u003c/sup\u003e group, seven belonged to the MIKC*, eight belonged to Ma, three to Mb, and four to Mg group (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The sequences of the identified proteins sequences are available in \u003cb\u003eSupplementary File S1\u003c/b\u003e. We could resolve putative orthologies of 20 \u003cem\u003eA. trichopoda\u003c/em\u003e genes to \u003cem\u003eA. thaliana\u003c/em\u003e MADS-Box genes with maximum likelihood phylogenetics of the full-length proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and reciprocal best hit (RBH), a sequence similarity-based method.\u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, MIKC\u003csup\u003eC\u003c/sup\u003e proteins are grouped into 13 subfamilies: AGL1(AG/SHP/STK), AGL16, AGL12, AGL7 (AP1/CAL/FUL), AGL6, AGL4/9 (SEP), AP3/PI, AGL70 (MAF/FYF), AGL20 (SOC1), AGL22 (SVP), AGL32, AGL15, and one subfamily with three \u003cem\u003eAmborella\u003c/em\u003e proteins but no \u003cem\u003eArabidopsis\u003c/em\u003e protein, placed in a clade ancestral to the AGL12, CAL, AGL13, and SEP subfamilies. There were no \u003cem\u003eAmborella\u003c/em\u003e proteins in the MAF subfamily. Interestingly, an entire subfamily of MIKC* genes present in the \u003cem\u003eAmborella\u003c/em\u003e genome was absent in the \u003cem\u003eArabidopsis\u003c/em\u003e genome.\u003c/p\u003e \u003cp\u003eAll floral MADS-Box, A, B-, C- and E-function, orthologs were present in the \u003cem\u003eA. trichopoda\u003c/em\u003e genome, represented by one \u003cem\u003eAGL7/8/10/\u003c/em\u003e79 \u003cem\u003e(AmtrAGL7/8/10/\u003c/em\u003e79), two \u003cem\u003eAPETALLA3\u003c/em\u003e (\u003cem\u003eAmtrAP3-1\u003c/em\u003e and \u003cem\u003e\u0026minus;\u0026thinsp;2\u003c/em\u003e), two \u003cem\u003ePISTILLATA (AmtrPI-1\u003c/em\u003e and \u003cem\u003eAmtrPI-2\u003c/em\u003e), one \u003cem\u003eAGAMOUS\u003c/em\u003e (\u003cem\u003eAmtrAG\u003c/em\u003e), and two \u003cem\u003eSEPALLATA\u003c/em\u003e orthologs (\u003cem\u003eAmtrAGL4\u003c/em\u003e and \u003cem\u003e9\u003c/em\u003e), respectively. Any close D-function orthologs (\u003cem\u003eAGL11\u003c/em\u003e), were not predicted by our methods, although \u003cem\u003eAmTrH2.11G126900.1.p\u003c/em\u003e did nest in the \u003cem\u003eAG/SHP/STK\u003c/em\u003e clade.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eClassification and nomenclature of \u003cem\u003eAmborella trichopoda\u003c/em\u003e MADS-box proteins based on orthologous relationships inferred from the Maximum Likelihood tree (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and the Reciprocal Best Hit (RBH) analysis (\u003cb\u003eSupplementary File S2\u003c/b\u003e) Sequences from \u003cem\u003eA. trichopoda\u003c/em\u003e that lack a putative ortholog \u003cem\u003ein Arabidopsis thaliana\u003c/em\u003e are designated as N/A.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA. trichopoda\u003c/em\u003e Protein\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eClosest \u003cem\u003eA. thaliana\u003c/em\u003e Ortholog\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOrtholog-based Name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eClass\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmTrH2.01G136300.1.p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAmtrAG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMIKC\u003csup\u003eC\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmTrH2.11G127800.1.p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAGL12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAmtrAGL12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMIKC\u003csup\u003eC\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmTrH2.01G083000.1.p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAGL15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAmtrAGL15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMIKC\u003csup\u003eC\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmTrH2.13G099300.1.p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAGL16/44/17/21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAmtrAGL16/44/17/21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMIKC\u003csup\u003eC\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmTrH2.04G078400.1.p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAGL22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAmtrAGL22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMIKC\u003csup\u003eC\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmTrH2.09G085100.1.p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAGL32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAmtrAGL32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMIKC\u003csup\u003eC\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmTrH2.10G036200.1.p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAGL4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAmtrAGL4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMIKC\u003csup\u003eC\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmTrH2.09G080800.1.p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAGL6/13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAmtrAGL6/13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMIKC\u003csup\u003eC\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmTrH2.10G037100.1.p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAGL7/8/10/79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAmtrAGL7/8/10/79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMIKC\u003csup\u003eC\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmTrH2.06G043000.1.p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAGL9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAmtrAGL9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMIKC\u003csup\u003eC\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmTrH2.06G157000.1.p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAP3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAmtrAP3-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMIKC\u003csup\u003eC\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmTrH2.09G064400.1.p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAP3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAmtrAP3-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMIKC\u003csup\u003eC\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmTrH2.06G101000.1.p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAmtrPI-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMIKC\u003csup\u003eC\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmTrH2.11G096800.1.p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAmtrPI-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMIKC\u003csup\u003eC\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmTrH2.05G000900.1.p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMIKC\u003csup\u003eC\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmTrH2.06G043400.1.p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMIKC\u003csup\u003eC\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmTrH2.06G043600.1.p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMIKC\u003csup\u003eC\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmTrH2.09G064500.1.p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMIKC\u003csup\u003eC\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmTrH2.09G080700.1.p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAGL20/42/71/72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAmtrAGL20/42/71/72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMIKC\u003csup\u003eC\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmTrH2.11G126900.1.p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMIKC\u003csup\u003eC\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmTrH2.04G150100.1.p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAGL66/67/104\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAmtrAGL66/67/104\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMIKC*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmTrH2.04G179300.1.p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAGL30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAmtrAGL30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMIKC*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmTrH2.04G099600.1.p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMIKC*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmTrH2.08G064100.1.p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMIKC*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmTrH2.09G031900.1.p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMIKC*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmTrH2.09G032000.1.p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMIKC*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmTrH2.10G045800.1.p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMIKC*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmTrH2.01G037700.1.p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAGL80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAmtrAGL80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMg\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmTrH2.03G113500.1.p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMg\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmTrH2.04G040100.1.p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMg\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmTrH2.11G086700.1.p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMg\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmTrH2.03G034800.1.p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAGL103\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAmtrAGL103\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMb\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmTrH2.01G056400.1.p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMb\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmTrH2.01G174800.1.p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMb\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmTrH2.01G163400.1.p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAGL62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAmtrAGL62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMa\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmTrH2.01G096900.1.p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMa\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmTrH2.01G097000.1.p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMa\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmTrH2.01G097100.1.p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMa\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmTrH2.07G063700.1.p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMa\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmTrH2.07G063800.1.p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMa\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmTrH2.07G084400.1.p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMa\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmTrH2.13G009700.1.p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMa\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePhysicochemical Properties and Sub-Cellular Localization\u003c/h2\u003e \u003cp\u003eAs shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, the longest and largest MADS-Box protein in \u003cem\u003eA. trichopoda\u003c/em\u003e was predicted to be AmtrAGL103 (Mb class), with a length of 378 amino acids (aa) and molecular weight (MW) of 43318.49 Daltons (Da). It was followed by AmtrAGL66/67/104 (MIKC*) which was 359 aa long and had a MW of 40813.66 Da. The shortest MADS-Box protein was predicted to be AmTrH2.04G099600.1.p with 99aa in length and 11539.68 Da in MW. Only nine of 42 MADS-Box proteins were predicted to have acidic isoelectric points (pI\u0026thinsp;\u0026lt;\u0026thinsp;6).\u003c/p\u003e \u003cp\u003eConsistent with the nature of transcription factors, 33 of the 42 identified MADS-Box proteins were predicted to localize in the nucleus, while the remaining nine were chloroplast-localized, albeit with scores below 1.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e, Summary of Predicted Physicochemical Properties and subcellular localization of \u003cem\u003eA. trichopoda\u003c/em\u003e MADS-Box proteins.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMADS-Box Protein\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLength (aa)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTheoretical Isoelectric Points (pI)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMolecular Weight (Daltons)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePredicted Subcellular Localization\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmtrAG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e235\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e26978.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmtrAGL12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e204\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e23277.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmtrAGL15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e257\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e29120.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmtrAGL16/44/17/21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e273\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e31351.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmtrAGL22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e221\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e25144.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmtrAGL32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e231\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e26867.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmtrAGL4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e243\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e27832\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmtrAGL6/13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e266\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e30379.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmtrAGL7/8/10/79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e242\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e28349.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmtrAGL9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e241\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e27357\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmtrAP3-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e220\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e26079.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmtrAP3-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e221\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e26166.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmtrPI-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e212\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24277.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmtrPI-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e211\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24725.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eChloroplast\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmTrH2.05G000900.1.p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e257\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e29519.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eChloroplast\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmTrH2.06G043400.1.p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e188\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e22018.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmTrH2.06G043600.1.p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e216\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e25246.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eChloroplast\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmTrH2.09G064500.1.p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e210\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24276.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmtrAGL20/42/71/72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e243\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e27541.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmTrH2.11G126900.1.p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e222\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e25650\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmtrAGL66/67/104\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e359\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e40813.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmtrAGL30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e347\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e39375.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmTrH2.04G099600.1.p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11539.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eChloroplast\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmTrH2.08G064100.1.p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e108\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12947.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eChloroplast\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmTrH2.09G031900.1.p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e304\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e34061.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmTrH2.09G032000.1.p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e186\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e22020.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eChloroplast\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmTrH2.10G045800.1.p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e335\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e38133.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmtrAGL80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e228\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e25602.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmTrH2.03G113500.1.p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e106\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12218.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eChloroplast\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmTrH2.04G040100.1.p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e355\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e39804.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmTrH2.11G086700.1.p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e144\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16633.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmtrAGL103\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e378\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e43318.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmTrH2.01G056400.1.p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e199\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e22541.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmTrH2.01G174800.1.p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e328\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e38151.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmtrAGL62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e212\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e23550.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmTrH2.01G096900.1.p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e211\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24405.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmTrH2.01G097000.1.p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e189\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e21539.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eChloroplast\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmTrH2.01G097100.1.p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e211\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e23924.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eChloroplast\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmTrH2.07G063700.1.p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16873.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmTrH2.07G063800.1.p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e262\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e29351.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmTrH2.07G084400.1.p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e367\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e41216.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmTrH2.13G009700.1.p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e153\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16793.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eConserved Motif Composition of\u003c/b\u003e \u003cb\u003eA. trichopoda\u003c/b\u003e \u003cb\u003eMADS-Box proteins\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAnalysis of conserved motifs in \u003cem\u003eA. trichopoda\u003c/em\u003e MADS-Box proteins showed distinct class-specific motif compositions as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Most MIKC-type proteins had similar motif composition in their M-domains (Motifs 2, 1, and 4), with some exceptions. All MIKC\u003csup\u003eC\u003c/sup\u003e-type proteins were characterized by the presence of the K-domain (Motif 3), while MIKC* proteins lacked the K-domain and had a slightly shorter motif signature (Motif 6) instead of the K-domain. All but one Mg protein (AmtrAGL80) lacked the K-domain, despite most of them having the 2, 1, and 4 motifs representing the M-domain. Motifs 5 and 7 were unique to Ma proteins, while Motif 8 was seen in Mg and Mb proteins, and interestingly, the two PI orthologs (AmtrPI-1 and \u0026minus;\u0026thinsp;2). Motif 10, which differs slightly from Motif 2, was unique to Mg proteins. Conserved Motif 9 was present in the C-terminal regions of the two SEPALLATA othologs (AmtrAGL-4 and \u0026minus;\u0026thinsp;9) and AmtrAGL6/13, proteins that nested in the same sub-family in our ML tree (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eStructure Analysis of\u003c/b\u003e \u003cb\u003eA. trichopoda\u003c/b\u003e \u003cb\u003eMADS-Box Genes\u003c/b\u003e\u003c/p\u003e \u003cp\u003eStructure analysis of MADS-Box genes in \u003cem\u003eA. trichopoda\u003c/em\u003e revealed class-specific exon-intron composition patterns (see Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). MIKC* and MIKC\u003csup\u003eC\u003c/sup\u003e-type genes were characterized by multiple exons. The first exon in all Type-II MADS-Box genes corresponded to the one encoding the M-domain.\u003c/p\u003e \u003cp\u003e \u003cem\u003eAmTrH2.06G043600.1.p\u003c/em\u003e was the longest MADS-Box gene in \u003cem\u003eA. trichopoda\u003c/em\u003e at 79,504 bp long, while the shortest one was \u003cem\u003eAmTrH2.03G113500.1.p\u003c/em\u003e (504bp). The B-function gene orthologs (\u003cem\u003eAmtrAP3-1\u003c/em\u003e and \u003cem\u003e\u0026minus;\u0026thinsp;2\u003c/em\u003e; \u003cem\u003eAmtrPI-1\u003c/em\u003e and \u003cem\u003e\u0026minus;\u0026thinsp;2\u003c/em\u003e) were among the shortest genes in the Type-II group, with \u003cem\u003eAmTrH2.09G064500.1.p\u003c/em\u003e being the other \u003cem\u003eAmtrAGL66/67/104\u003c/em\u003e had the greatest number of exons (11), while many Type-I MADS-Box genes had a single exon. \u003cem\u003eAmtrAGL103\u003c/em\u003e\u0026rsquo;s single exon was the longest in all MADS-Box genes (1137bp).\u003c/p\u003e \u003cp\u003eDetails of gene structural attributes are summarized in \u003cb\u003eSupplementary File S3\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCis-Regulatory Elements (CREs) of\u003c/b\u003e \u003cb\u003eA. trichopoda\u003c/b\u003e \u003cb\u003eMADS-Box Genes\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAltogether 104 distinct \u003cem\u003ecis\u003c/em\u003e-regulatory elements were predicted in the 2000bp upstream regions of the 42 \u003cb\u003eA. trichopoda\u003c/b\u003e MADS-Box genes. The co-ordinates of the 20 most prevalent CREs are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. With an average occurrence per sequence of 44.90, TATA-Box was the most common cis-element, followed by CAAT-Box (35.33). The CTCC motif followed occurring at an average of 10.57 times per gene. A complete list of all identified \u003cem\u003ecis\u003c/em\u003e-element in the 2000bp upstream regions of MADS-Box genes and their co-ordinates are summarized in \u003cb\u003eSupplementary File S4\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eGene Ontology\u003c/h3\u003e\n\u003cp\u003eGene Ontology analysis of the identified MADS-Box proteins returned 22 GO terms corresponding to their predicted functions, as visualized in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. Under the \u0026ldquo;Cellular Component\u0026rdquo; ontology, \u0026ldquo;Nucleus\u0026rdquo; had the greatest number of hits (35), \u0026ldquo;Molecular Function\u0026rdquo; ontology had \u0026ldquo;DNA-binding transcription factor activity, RNA polymerase II-specific\u0026rdquo;, and \u0026ldquo;protein dimerization activity\u0026rdquo; had 35 hits each. Under the \u0026ldquo;Biological Process\u0026rdquo; ontology, \u0026ldquo;regulation of transcription by RNA polymerase II\u0026rdquo; had the highest number of hits (20), followed by \u0026ldquo;positive regulation of transcription by RNA polymerase II\u0026rdquo; (16). All these assigned ontologies are consistent with MADS-Box proteins being transcription factors that form multimers and bind to DNA.\u003c/p\u003e \u003cp\u003e \u003cb\u003eChromosomal Locations of MADS-Box Genes in\u003c/b\u003e \u003cb\u003eA. trichopoda\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e shows MADS-Box genes mapped to 11 of the 13 chromosomes of \u003cem\u003eA. trichopoda\u003c/em\u003e, with none located on chromosomes 2 and 12. Chromosome 1 had the highest concentration of MADS-Box genes (nine in total) while chromosomes 5 and 8 had one each. All MADS-Box genes on chromosome 6 were MIKC\u003csup\u003eC\u003c/sup\u003e-type, while three of five MADS-Box genes on chromosome 4 were MIKC*-type. Chromosomes 9 and 10 exclusively contained MIKC-type genes while chromosome 7 contained only Ma genes.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEvolutionary Selection Pressure in\u003c/b\u003e \u003cb\u003eAmborella trichopoda\u003c/b\u003e \u003cb\u003eMADS-Box genes (Ka/Ks analysis)\u003c/b\u003e\u003c/p\u003e \u003cp\u003eA Ka/Ks analysis of \u003cem\u003eA. trichopoda\u003c/em\u003e MADS-Box genes revealed an average Ka/Ks value of 0.308 among all possible gene pair combinations, suggesting a strong purifying selection among these genes. The only paralogous gene pairs with a Ka/Ks value of more than one (\u0026gt;\u0026thinsp;1) were \u003cem\u003eAmTrH2.01G096900.1/AmTrH2.01G097000.1\u003c/em\u003e and \u003cem\u003eAmTrH2.01G097100.1/ AmTrH2.01G097000.1\u003c/em\u003e with Ka/Ks values of 1.08 and 1.07 respectively. All these genes belong to the Type I (Ma) group. Ka/Ks values of all gene pairs are provided in \u003cb\u003eSupplementary File S5\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCollinearity of\u003c/b\u003e \u003cb\u003eA. trichopoda\u003c/b\u003e \u003cb\u003eMADS-Box Genes with\u003c/b\u003e \u003cb\u003eA. thaliana\u003c/b\u003e \u003cb\u003eMADS-Box Genes\u003c/b\u003e\u003c/p\u003e \u003cp\u003eOut of 85230 total genes in both species, 1790 (2.10%) formed collinear blocks (defined as five consecutive genes) between the genomes. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, four MADS-Box genes in \u003cem\u003eAmborella\u003c/em\u003e were found in collinear blocks with \u003cem\u003eArabidopsis\u003c/em\u003e, two of which (\u003cem\u003eAmtrAGL30\u003c/em\u003e and \u003cem\u003eAmtrAGL66/67/104\u003c/em\u003e) were of the MIKC*-type, and were located on chromosome 4. Also, \u003cem\u003eAmtrAGL7/8/10/79\u003c/em\u003e was collinear with \u003cem\u003eArabidopsis AGL7\u003c/em\u003e and \u003cem\u003eAmtrAGL12\u003c/em\u003e was collinear with its \u003cem\u003eArabidopsis\u003c/em\u003e ortholog.\u003c/p\u003e \u003cp\u003eNo Type-I MADS-Box genes were found in collinear blocks between the two species.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDifferential Expression of\u003c/b\u003e \u003cb\u003eA. trichopoda\u003c/b\u003e \u003cb\u003eMADS-Box Genes in Male and Female Tissues\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTwenty MADS-Box genes were found to be differentially expressed between mature male and female flowers of \u003cem\u003eA. trichopoda\u003c/em\u003e (padj\u0026thinsp;\u0026le;\u0026thinsp;0.05), and all of them were Type-II MADS-Box genes. Twelve of these genes were downregulated while eight were upregulated in females.\u003c/p\u003e \u003cp\u003e \u003cem\u003eAmtrPI-2\u003c/em\u003e gene had the most significant downregulation in female flowers (padj\u0026thinsp;=\u0026thinsp;1.64e-134 and log2FC = -2.82) while the MIKC* gene \u003cem\u003eAmtrAGL66/67/104\u003c/em\u003e had the highest downregulation in terms of log2FC value in females compared to males (padj\u0026thinsp;=\u0026thinsp;2.55e-70 and log2FC = -7.48). Among the upregulated genes in mature female flowers, \u003cem\u003eAmtrAGL32\u003c/em\u003e had the highest upregulation (log2FC\u0026thinsp;=\u0026thinsp;7.26, padj\u0026thinsp;=\u0026thinsp;1.27e-27), followed by \u003cem\u003eAmTrH2.11G126900.1\u003c/em\u003e (log2FC\u0026thinsp;=\u0026thinsp;4.09, padj\u0026thinsp;=\u0026thinsp;1.84e-37). Two \u003cem\u003eAP3\u003c/em\u003e orthologs exhibited distinct expression pattern: \u003cem\u003eAmtrAP3-1\u003c/em\u003e was upregulated in female flowers while \u003cem\u003eAmtrAP3-2\u003c/em\u003e was upregulated in male flowers. The volcano plot in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e summarizes the differential expression of MADS-Box genes in mature \u003cem\u003eAmborella\u003c/em\u003e male and female flowers. Principal Component Analysis (PCA) of the normalized raw read counts in the male and female mature flowers showed a clear distinction between two sexes based on the expression of MADS-Box genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e, suggesting a distinct sex-specific expression pattern of MADS-Box genes in mature flowers of \u003cem\u003eAmborella\u003c/em\u003e. The heatmap in Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e shows expression patterns of MADS-Box genes with non-zero read counts in mature flowers of the two sexes with z-transformed Transcript per Million kilobase (TPM) values. The samples distinctly clustered according to their sex based on the expression patterns of MADS-Box genes in mature flowers.\u003c/p\u003e \u003cp\u003eIn floral buds, only seven MADS-Box genes were differentially expressed between male and female samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. Of them, four were downregulated in females: \u003cem\u003eAmTrH2.11G126900.1\u003c/em\u003e (log2FC = -1.05. padj\u0026thinsp;=\u0026thinsp;7.46e-07). \u003cem\u003eAmtrAGL32\u003c/em\u003e (log2FC value: -4.51, padj\u0026thinsp;=\u0026thinsp;0.0009) and \u003cem\u003eAmtrAGL15\u003c/em\u003e (log2FC = -0.55, padj\u0026thinsp;=\u0026thinsp;0.0005) in female buds. The three upregulated genes were \u003cem\u003eAmtrAP3-2\u003c/em\u003e, \u003cem\u003eAmtrPI-2\u003c/em\u003e, \u003cem\u003eAmTrH2.09G064500.1\u003c/em\u003e, and \u003cem\u003eAmTrH2.07G063800.1\u003c/em\u003e. Interestingly, \u003cem\u003eAmtrAP3-2\u003c/em\u003e and \u003cem\u003eAmtrPI-2\u003c/em\u003ewere downregulated in mature female flowers but are upregulated in female floral buds. Additionally, \u003cem\u003eAmTrH2.07G063800.1\u003c/em\u003e, an Ma gene, was the only Type-I MADS-Box gene differentially expressed between male and female floral buds. Principal Component Analysis (PCA) of normalized read counts of male and female floral buds had a lot of overlap between the 95% confidence interval ellipses (see Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003e), showing that unlike in mature flowers, the expression pattern of MADS-Box genes in floral buds of \u003cem\u003eAmborella\u003c/em\u003e is not highly sex-specific. Clustering of samples based on expression patterns (z-transformed normalized read counts) based on Euclidean distances could not cluster male and female floral buds distinctly, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e13\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eAll gene expression data are available in \u003cb\u003eSupplementary File S6\u003c/b\u003e.\u003c/p\u003e "},{"header":"Discussion","content":"\u003cp\u003e \u003cb\u003eAmborella trichopoda\u003c/b\u003e \u003cb\u003eMADS-Box Genes Identification and Nomenclature\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe identified 42 MADS-Box genes in \u003cem\u003eA. trichopoda\u003c/em\u003e which belonged to all the major classes of plant MADS-Box genes. We used a homology-based approach to name the genes, referencing their putative orthology with \u003cem\u003eA. thaliana\u003c/em\u003e MADS-Box genes. With the decreasing costs of Next-Generation Sequencing (NGS) technologies, there has been a dramatic increase in the number of plant genomes coming from research groups around the world. Public availability of such genomes has subsequently accelerated genome-wide identification studies of genes and gene families in plants, advancing our understanding of evolutionary processes, functional genomics, and plant adaptation to diverse environments. However, the procedure for naming genes in a gene family has not been consistent across different studies. For instance, the identified MADS-Box genes were named following their chromosomal locations in \u003cem\u003eZizania latifolia\u003c/em\u003e\u003csup\u003e\u003cem\u003e27\u003c/em\u003e\u003c/sup\u003e. In some cases, no specific naming criteria were applied as in the case of \u003cem\u003eGlycine max\u003c/em\u003e\u003csup\u003e28\u003c/sup\u003e, \u003cem\u003eOryza sativa\u003c/em\u003e\u003csup\u003e29\u003c/sup\u003e, and \u003cem\u003eMalus domestica\u003c/em\u003e\u003csup\u003e30\u003c/sup\u003e. MADS-Box genes were named after their gene ids in increasing order in \u003cem\u003eSesamum indicum\u003c/em\u003e\u003csup\u003e31\u003c/sup\u003e, and in two orchids: \u003cem\u003eDendrobium officinale\u003c/em\u003e and \u003cem\u003ePhalaenopsis equestris\u003c/em\u003e, the authors did not specify how the identified genes were named, even though the genes were assigned orthologies with their \u003cem\u003eA. thaliana\u003c/em\u003e counterparts\u003csup\u003e32\u003c/sup\u003e. These inconsistencies can present challenges not only in identifying genes, but also in assigning functions to these genes, as genes with the same number may not have the same functional annotation across species. A more consistent approach, such as assigning gene names based on verified or predicted biological functions inferred from sequence homology, would help standardize gene naming and facilitate cross-species comparisons. .\u003c/p\u003e \u003cp\u003eSuch standardizations in nomenclature have been suggested for multiple animal gene families\u003csup\u003e33\u0026ndash;36\u003c/sup\u003e. Efforts have been made to standardize nomenclature for plant gene families as well. Orthology-based nomenclature for plant WRKY genes was recommended by Mohanta et al. in 2016\u003csup\u003e37\u003c/sup\u003e. Naming guidelines have been proposed for rice WRKY\u003csup\u003e38\u003c/sup\u003e, \u003cem\u003eA. trichopoda\u003c/em\u003e WRKY\u003csup\u003e39\u003c/sup\u003e, plant HKT\u003csup\u003e38\u003c/sup\u003e, and MAPK\u003csup\u003e40\u003c/sup\u003e gene families. In the present study, we named each \u003cem\u003eAmborella trichopoda\u003c/em\u003e MADS-Box gene by following an orthology-based approach, where the first two letters of the gene name are derived from the genus and species names, followed by 'AGLx' (AGAMOUS-like and a number x), corresponding to the orthologous \u003cem\u003eA. thaliana\u003c/em\u003e gene. For instance, 'AmTrAGL1' refers to the AGL1 ortholog in \u003cem\u003eAmborella trichopoda\u003c/em\u003e. Since 'At' is already used to abbreviate \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, we used 'AmTr' (from the first two letters of the genus and species) to abbreviate \u003cem\u003eAmborella trichopoda\u003c/em\u003e.. Assigning orthologies based solely on sequence similarity, however, might lead to errors in functional annotation because of possible differences in expression patterns, for instance, AG and AGL1 have a very similar sequence structure but can have distinct expression patterns and functions\u003csup\u003e8\u003c/sup\u003e. Gene expression patterns, mutagenic and/or overexpression studies can thus provide the strongest evidence of orthology\u003csup\u003e9,41\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAlthough a detailed report on \u003cem\u003eA. trichopoda\u003c/em\u003e MADS-Box genes has not been published yet, previous studies have reported varying numbers of \u003cem\u003eAmborella\u003c/em\u003e MADS-Box genes: 36 in one study \u003csup\u003e24\u003c/sup\u003e and 33 in another \u003csup\u003e32\u003c/sup\u003e. With initial HMMER\u003csup\u003e42\u003c/sup\u003e searches, we obtained 46 unique hits in this study, four of which were filtered out because they lacked MADS domain, as confirmed by the Conserved Domain Database (CDD)\u003csup\u003e43\u003c/sup\u003e, Simple Modular Architecture Research Tool (SMART)\u003csup\u003e44\u003c/sup\u003e or Pfam\u003csup\u003e45\u003c/sup\u003e domain searches. In fact, The Arabidopsis Information Resource (TAIR)\u003csup\u003e46\u003c/sup\u003e\u0026rsquo;s \u003cem\u003eA. thaliana\u003c/em\u003e MADS-Box gene family entry from AGRIS had 109 gene entries at the time of writing this article, but three of those genes did not have the MADS domain as per our domain search and three did not have a predicted protein sequence, and hence were excluded from further analyses. Upon manual analysis of hits from our two HMMER searches, one with a Pfam HMM profile entry PF00319, and the other with an HMM profile generated from alignment of all \u003cem\u003eA. thaliana\u003c/em\u003e MADS-Box protein sequences, we could observe that all entries filtered from our domain confirmation were identified as hits from the first HMMER search itself, making the second HMMER search redundant. With this, we recommend that, if available, a HMMER search with the PFam HMM profile in the Interpro database would be sufficient to identify gene family homologs in a genome for the MADS-Box gene family, provided that the expect threshold (e-value) is not too selective, as it could potentially filter out distant homologs.\u003c/p\u003e \u003cp\u003e \u003cb\u003eClassification and Evolutionary Dynamics of\u003c/b\u003e \u003cb\u003eA. trichopoda\u003c/b\u003e \u003cb\u003eMADS-Box Genes\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAmong the 42 \u003cem\u003eA. trichopoda\u003c/em\u003e MADS-Box genes identified in this study, 20 genes were classified as MIKC\u003csup\u003eC\u003c/sup\u003e, seven as MIKC*, eight as Ma, three as Mb, and four as Mg. Different numbers of MADS-Box genes have been reported in various algae and plant species. No MIKC\u003csup\u003eC\u003c/sup\u003e or MIKC* genes were found in green algae, but a single gene with a MEF2-like M-domain without I, K, or C domains was reported in in \u003cem\u003eChlamydomonas reinhardtii\u003c/em\u003e and \u003cem\u003eC. merolae\u003c/em\u003e\u003csup\u003e15\u003c/sup\u003e. One MIKC type gene was reported in Charophycean algae \u003cem\u003eChara globularis, C. scutata\u003c/em\u003e, and \u003cem\u003eC. peracerosum-strigosum-littorale\u003c/em\u003e complex\u003csup\u003e17\u003c/sup\u003e, while 26 MADS-Box genes (17 MIKC and 9 M-type) were found in the bryophyte \u003cem\u003ePhyscomitrella patens\u003c/em\u003e\u003csup\u003e47\u003c/sup\u003e, 19 (6 MIKC and 13 M-type) in the lycophyte \u003cem\u003eSelaginella moellendorffii\u003c/em\u003e\u003csup\u003e48\u003c/sup\u003e, and 36 (35 MIKC and 1 M-type) in the fern \u003cem\u003eVandenboschia speciosa\u003c/em\u003e\u003csup\u003e49\u003c/sup\u003e. The presence of MIKC* genes in bryophytes suggests that at least one MIKC* gene was present in the common ancestor of bryophytes and seedless vascular plants, likely evolving from an MIKC\u003csup\u003eC\u003c/sup\u003e-type gene (reviewed in\u003csup\u003e47\u003c/sup\u003e). Despite MIKC\u003csup\u003eC\u003c/sup\u003e genes being present in seedless plants, they are not orthologous to phanerogamic (gymnosperm or angiosperm) MIKC\u003csup\u003eC\u003c/sup\u003e genes, hinting at independent evolution of these genes in these plant lineages\u003csup\u003e12\u003c/sup\u003e. MIKC* genes, on the other hand, show considerable homology and conservation of function across seedless plants, gymnosperms and angiosperms (reviewed in\u003csup\u003e47\u003c/sup\u003e).\u003c/p\u003e \u003cp\u003eThe number of MADS-Box genes varies greatly in gymnosperms ranging from three (all MIKC) in \u003cem\u003eTaxus baccata\u003c/em\u003e to 367 (350 MIKC and 17 M-type) in \u003cem\u003ePinus taeda\u003c/em\u003e\u003csup\u003e50\u003c/sup\u003e. In our study, we classified \u003cem\u003eA. trichopoda\u003c/em\u003e MIKC\u003csup\u003eC\u003c/sup\u003e genes into 12 subfamilies: AGL1(AG/SHP/STK), AGL16, AGL12, AGL7 (AP1/CAL/FUL), AGL6, AGL4/9 (SEP), AP3/PI, AGL20 (SOC1), AGL22 (SVP), AGL32, AGL15, and a clade ancestral to the AGL12, CAL, AGL13, and SEP subfamilies, specific to \u003cem\u003eAmborella\u003c/em\u003e. The MAF/FLC and FYF clades represented in \u003cem\u003eA. thaliana\u003c/em\u003e were however absent in \u003cem\u003eA. trichopoda\u003c/em\u003e. Our results agree largely with that of a previous study that identified 14 MIKC\u003csup\u003eC\u003c/sup\u003e clades in gymnosperms and basal angiosperms: \u003cem\u003eSVP, MADS32, AP3/PI, AGL32, AGL15, AG, ANR1, AGL12, SOC1, GMADS, FLC, AP1/FUL, AGL6\u003c/em\u003e, and \u003cem\u003eSEP\u003c/em\u003e\u003csup\u003e51\u003c/sup\u003e, except for GMADS, which is gymnosperm-specific; \u003cem\u003eMADS32\u003c/em\u003e, which is a clade with \u003cem\u003eOsMADS32\u003c/em\u003e from rice, a species not included in our study, and \u003cem\u003eMAF/FLC\u003c/em\u003e: a group found only in Asteraceae\u003csup\u003e52,53\u003c/sup\u003e, the botanical family of \u003cem\u003eA. thaliana\u003c/em\u003e. Our results are largely similar to a recent study that reported 13 subfamilies of \u003cem\u003eAmborella\u003c/em\u003e MIKC\u003csup\u003eC\u003c/sup\u003e genes, and identified single orthologs in \u003cem\u003ePI\u003c/em\u003e and \u003cem\u003eAG\u003c/em\u003e groups\u003csup\u003e26\u003c/sup\u003e. Our results confirm a previous report that only a single copy of \u003cem\u003eAP1/FUL/SOC1\u003c/em\u003e genes is present in \u003cem\u003eA. trichopoda\u003c/em\u003e\u003csup\u003e51\u003c/sup\u003e. Absent in gymnosperms, the \u003cem\u003eAP1/FUL\u003c/em\u003e family first appeared in angiosperms\u003csup\u003e54\u003c/sup\u003e, diverging into two unique groups in monocots and three groups in eudicots\u003csup\u003e51\u003c/sup\u003e. The presence of only one gene (name) in the \u003cem\u003eAP1/FUL\u003c/em\u003e family in \u003cem\u003eA. trichopoda\u003c/em\u003e that shared orthology to \u003cem\u003eAGL- 7, 10, 9\u003c/em\u003e, and \u003cem\u003e79\u003c/em\u003e in our study suggests that \u003cem\u003eAP1\u003c/em\u003e (\u003cem\u003eAGL7)\u003c/em\u003e-specific orthologs are probably not present in \u003cem\u003eA. trichopoda\u003c/em\u003e and evolved later in the angiosperm lineage. This result aligns with a previous report that that \u003cem\u003eeuAP1\u003c/em\u003e may have originated from a frameshift mutation in an ancient \u003cem\u003eeuFUL-\u003c/em\u003e or \u003cem\u003eFUL-like\u003c/em\u003e gene\u003csup\u003e54\u003c/sup\u003e. This absence of a direct \u003cem\u003eAGL7\u003c/em\u003e ortholog and statistically similar expression of the ancestral ortholog in both male and female flowers, discussed later, suggests the possibility of this gene carrying out the A-function or its complete absence in \u003cem\u003eA. trichopoda\u003c/em\u003e, possibly explaining the absence of differentiated calyx and corolla whorls in its flowers. Orthologs of \u003cem\u003eAP2\u003c/em\u003e, a non-MADS-Box A-function gene, were found in \u003cem\u003eA. trichopoda\u003c/em\u003e (blast search, data not shown), however, \u003cem\u003eAmtrAGL7/8/10/79\u003c/em\u003e was one of the four MADS-Box genes in \u003cem\u003eAmborella\u003c/em\u003e \u0026ndash; present in a genomic collinear block with \u003cem\u003eArabidopsis\u003c/em\u003e, suggesting a strong conservation of function of these genes across plant lineages.\u003c/p\u003e \u003cp\u003eIn \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, B-function is controlled by the \u003cem\u003eAP3\u003c/em\u003e and \u003cem\u003ePI\u003c/em\u003e genes\u003csup\u003e55\u003c/sup\u003e. We found two orthologs for each in \u003cem\u003eAmborella trichopoda\u003c/em\u003e: \u003cem\u003eAmtrAP3-1, AmtrAP3\u003c/em\u003e-\u003cem\u003e2, AmtrPI-1\u003c/em\u003e and \u003cem\u003eAmtrPI-2\u003c/em\u003e. These genes were placed in \u003cem\u003eA. trichopoda\u003c/em\u003e\u0026rsquo;s \u003cem\u003eAP3/PI\u003c/em\u003e group in a previous study\u003csup\u003e51\u003c/sup\u003e, but the two \u003cem\u003eAP3\u003c/em\u003e orthologs were not named as such. \u003cem\u003eAP3\u003c/em\u003e orthologs have been identified in gymnosperms: \u003cem\u003ePrDGL\u003c/em\u003e in \u003cem\u003ePinus radiata\u003c/em\u003e\u003csup\u003e56\u003c/sup\u003e, \u003cem\u003eGGM13\u003c/em\u003e and \u003cem\u003eGGM2\u003c/em\u003e in \u003cem\u003eGnetum gnemon\u003c/em\u003e\u003csup\u003e56\u003c/sup\u003e, and DEFICIENS-AGAMOUS-LIKE (DAL) \u003cem\u003e\u0026minus;\u0026thinsp;11, 12\u003c/em\u003e, and \u003cem\u003e13\u003c/em\u003e in \u003cem\u003ePicea abies\u003c/em\u003e\u003csup\u003e57\u003c/sup\u003e. The B-function of specifying male reproductive organ identity is conserved in conifer DEF/GLO-like proteins, but in contrast, \u003cem\u003eGGM13\u003c/em\u003e-like genes is expressed preferentially in female tissues\u003csup\u003e50\u003c/sup\u003e, also known as B-sister MADS-Box genes\u003csup\u003e58\u003c/sup\u003e. Since \u003cem\u003eAmtrAP3-1\u003c/em\u003e is not related to \u003cem\u003eGGM13\u003c/em\u003e in our phylogenetic analysis, it is not a B-sister MADS-Box gene. Instead, one \u003cem\u003eA. trichopoda\u003c/em\u003e protein AmTrH2.09G085100.1.p (AmtrAGL32) nested in the AGL32 (\u003cem\u003eArabidopsis\u003c/em\u003e B-sister) clade in our tree. Expression data (discussed later) showed upregulation of this gene in mature female flowers, suggesting potential conservation of function of B-sister MADS-Box genes in early angiosperms and gymnosperms. Since \u003cem\u003eGGM13\u003c/em\u003e was already present in gymnosperms, we infer that the \u003cem\u003eA. trichopoda\u003c/em\u003e B-sister gene is a descendant of gymnosperm B-sister MADS-Box genes and likely existed before the divergence of angiosperms and gymnosperms. The \u003cem\u003eAmtrAP3-1\u003c/em\u003e gene was longer than \u003cem\u003eAmtrAP3-2\u003c/em\u003e, but the proteins they encode were nearly equal lengths, with the gene length difference attributed to intron size. Both proteins had nearly identical sequences and were characterized by the \u0026ldquo;DLRLG\u0026rdquo; motif at the C-terminal end, a signature paleoAP3 motif found in AP3 proteins in lower angiosperms. This supports the idea that the paleoAP3 motif is the common ancestral form of the euAP3 and TM6 lineages found in higher eudicots. Duplication in a paleoAP3 ancestor gave rise to the new lineages, and this event occurred after the common ancestor of Buxaceae and higher eudicots, but not later than the diversification into higher eudicot classes\u003csup\u003e59\u003c/sup\u003e. \u003cem\u003eA. trichopoda\u003c/em\u003e also has a distinct PI lineage, consisting of two PI orthologs. The PI lineage was probably formed in the lineage leading to angiosperms as a result of a duplication in the B-function gene lineage, of which elimination of the paleoAP3 motif was a major change in the new lineage\u003csup\u003e59\u003c/sup\u003e. AP3 and PI proteins share striking sequence similarity, except for the characteristic residues especially concentrated at the C-terminal motifs\u003csup\u003e60\u003c/sup\u003e. We are the first to report the presence of two PI orthologs in \u003cem\u003eA. trichopoda\u003c/em\u003e genome.\u003c/p\u003e \u003cp\u003eC-function floral MADS-Box genes specify stamen and carpel identity and are represented by \u003cem\u003eAGAMOUS\u003c/em\u003e (\u003cem\u003eAG\u003c/em\u003e), \u003cem\u003eSHATTERPROOF 1\u003c/em\u003e (\u003cem\u003eSHP1\u003c/em\u003e/\u003cem\u003eAGL1\u003c/em\u003e) and \u003cem\u003eSHATTERPROOF 2\u003c/em\u003e (\u003cem\u003eSHP2/AGL5\u003c/em\u003e), while D-function genes specify ovule identity, represented by \u003cem\u003eAGL11\u003c/em\u003e (\u003cem\u003eSTK\u003c/em\u003e) in \u003cem\u003eA. thaliana\u003c/em\u003e (reviewed in\u003csup\u003e61\u003c/sup\u003e). In \u003cem\u003eA. trichopoda\u003c/em\u003e, the \u003cem\u003eAG/SHP/STK\u003c/em\u003e clade had two genes: 1) \u003cem\u003eAmTrH2.01G136300\u003c/em\u003e, an \u003cem\u003eA. thaliana AG\u003c/em\u003e ortholog, hence named \u003cem\u003eAmtrAG\u003c/em\u003e and 2) \u003cem\u003eAmTrH2.11G126900\u003c/em\u003e nested in the \u003cem\u003eAG/SHP/STK\u003c/em\u003e clade, and the fact that it was upregulated in female buds and mature female flowers, suggests that it may be a D-function ortholog. The expression of \u003cem\u003eAmtrAG\u003c/em\u003e in both male and female flowers further supports our orthology assignment to \u003cem\u003eA. thaliana AG\u003c/em\u003e gene. C-function MADS-Box gene orthologs have been found in all seed plants, but not in non-seed plants\u003csup\u003e61\u003c/sup\u003e. Members of the \u003cem\u003eAGAMOUS\u003c/em\u003e clade are most likely the result of whole genome duplication events, the first of which probably occurred before the common ancestor of all extant angiosperms\u003csup\u003e62\u003c/sup\u003e, giving rise to C- and D-function genes with specialized functions\u003csup\u003e63\u003c/sup\u003e. Since \u003cem\u003eAmborella\u003c/em\u003e is a basal angiosperm and contains only two genes in the \u003cem\u003eAGAMOUS\u003c/em\u003e subfamily, one of each C- and D-function, it is highly probable that at least one copy of C- and D-function genes were present in the most basal angiosperm, which got expanded in higher eudicots probably because of gene and/or genome duplication events.\u003c/p\u003e \u003cp\u003eThe E-function of floral organ identity in \u003cem\u003eArabidopsis\u003c/em\u003e is carried out by \u003cem\u003eAGL2\u003c/em\u003e-like genes: AGL2, AGL3, AGL4, and AGL9\u003csup\u003e64\u0026ndash;67\u003c/sup\u003e. The \u003cem\u003eA. trichopoda\u003c/em\u003e genome has two \u003cem\u003eAGL2-\u003c/em\u003elike genes: \u003cem\u003eAmTrH2.10G036200\u003c/em\u003e and \u003cem\u003eAmTrH2.06G043000\u003c/em\u003e, which we named \u003cem\u003eAmtrAGL4 (AmtrSEP2)\u003c/em\u003e and \u003cem\u003eAmtrAGL9 (AmtrSEP3)\u003c/em\u003e, respectively based on our orthology assessment. Two \u003cem\u003eSEPALLATA (SEP)\u003c/em\u003e homologs were identified in \u003cem\u003eAmborella\u003c/em\u003e and named \u003cem\u003eAmtrAGL2 (SEP1)\u003c/em\u003e and \u003cem\u003eAmtrAGL9 (SEP3)\u003c/em\u003e in a previous study\u003csup\u003e68\u003c/sup\u003e. An analysis of \u003cem\u003eSEP\u003c/em\u003e homologs across a wide range of taxa showed that \u003cem\u003eSEP1\u003c/em\u003e and \u003cem\u003eSEP2\u003c/em\u003e homologs are restricted to Brassicaceae, while \u003cem\u003eSEP4\u003c/em\u003e is present only in core eudicots\u003csup\u003e69\u003c/sup\u003e. While our results also do not show the presence of \u003cem\u003eSEP1\u003c/em\u003e and \u003cem\u003eSEP4\u003c/em\u003e orthologs in \u003cem\u003eAmborella\u003c/em\u003e, the presence of a close \u003cem\u003eSEP2\u003c/em\u003e ortholog does not align with the previous findings. Only one \u003cem\u003eAGL2-\u003c/em\u003elike gene (\u003cem\u003ePRMADS1\u003c/em\u003e) was found in \u003cem\u003ePinus radiata\u003c/em\u003e\u003csup\u003e70\u003c/sup\u003e. However, since no other gymnosperm was found to have an \u003cem\u003eAGL2\u003c/em\u003e homolog, and \u003cem\u003ePRMADS1\u003c/em\u003e nested with an \u003cem\u003eEucalyptus AGL2-\u003c/em\u003elike gene in a phylogenetic tree, caution was advised when considering PRMADS1 as an AGL2 homolog\u003csup\u003e61\u003c/sup\u003e. Other basal angiosperms like Magnoliids and the basal eudicot \u003cem\u003eEschscholzia californica\u003c/em\u003e were also found to have two \u003cem\u003eSEP\u003c/em\u003e homologs\u003csup\u003e68\u003c/sup\u003e. These observations suggest that \u003cem\u003eSEP\u003c/em\u003e genes may have first originated in the common ancestor of all angiosperms. The \u003cem\u003eSEP\u003c/em\u003e clade is considered to be sister to the \u003cem\u003eAGL6\u003c/em\u003e clade\u003csup\u003e61\u003c/sup\u003e, which contains \u003cem\u003eAGL6\u003c/em\u003e and \u003cem\u003eAGL13\u003c/em\u003e from \u003cem\u003eArabidopsis\u003c/em\u003e. \u003cem\u003eAGL6\u003c/em\u003e homologs are also found in gymnosperms\u003csup\u003e61\u003c/sup\u003e. Our analysis revealed one \u003cem\u003eAmborella\u003c/em\u003e gene belonging to the \u003cem\u003eAGL6\u003c/em\u003e clade, and the \u003cem\u003eAGL6\u003c/em\u003e, \u003cem\u003eSEP\u003c/em\u003e, and \u003cem\u003eAP1/CAL/FUL\u003c/em\u003e clades formed a superclade. Sister clades \u003cem\u003eAP1/FUL/SQUA\u003c/em\u003e and \u003cem\u003eAGL6-SEP\u003c/em\u003e arose due to duplication events during evolution, of which only the \u003cem\u003eAGL6\u003c/em\u003e subfamily was retained in gymnosperms, while angiosperms retained the other subfamilies as well\u003csup\u003e71\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003eA. trichopoda\u003c/em\u003e has seven MIKC*-type genes, two of which share sequence homology with \u003cem\u003eArabidopsis\u003c/em\u003e MIKC* genes: 1) \u003cem\u003eAmTrH2.04G150100\u003c/em\u003e is now named \u003cem\u003eAmtrAGL66/67/104\u003c/em\u003e, and 2) \u003cem\u003eAmTrH2.04G179300\u003c/em\u003e is named \u003cem\u003eAmtrAGL30\u003c/em\u003e in the present study. Four MIKC* genes were first reported in the moss \u003cem\u003ePhyscomitrella patens\u003c/em\u003e, and they differ from MIKC\u003csup\u003eC\u003c/sup\u003e genes by the presence of a longer I-domain and have variable length and hydrophobic residues in the K-domain\u003csup\u003e12\u003c/sup\u003e. An elongation of the I-domain in the ancestral MIKC\u003csup\u003eC\u003c/sup\u003e-type gene was proposed to have given rise to the MIKC* lineage\u003csup\u003e12\u003c/sup\u003e, however later studies suggested that the MIKC* lineage was formed from a duplication in the region encoding the K-domain\u003csup\u003e72\u003c/sup\u003e. MIKC* genes have since been identified across all green plant lineages, from mosses to eudicots, and they are highly conserved in both structure and function from ferns to seed plants, although they are fewer in number in all plants compared to their MIKC\u003csup\u003eC\u003c/sup\u003e counterparts. It has been suggested that in most plant lineages, the MIKC* group contains two monophyletic clades, S and P, the origin of which can be traced back to more than 380 MYA to the ancestor of ferns and seed plants\u003csup\u003e73\u003c/sup\u003e. In the present study, the MIKC* subclade could be divided into two groups that had genes orthologous to \u003cem\u003eArabidopsis\u003c/em\u003e MIKC* genes, and a subclade that contained only \u003cem\u003eA. trichopoda\u003c/em\u003e genes. Interestingly, only \u003cem\u003eAmTrH2.08G064100.1\u003c/em\u003e had a non-zero read count in mature flowers, without differential expression between the sexes, suggesting that these genes may have escaped identification. Unlike other MIKC*-genes, they do not appear to be expressed in the male gametophyte, or they could potentially be pseudogenes. The apparent lack of 5\u0026rsquo; and 3\u0026rsquo; untranslated regions (UTRs) in these genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) supports the possibility that they could in-fact be pseudogenes. Also, the presence of two MIKC*-type genes: \u003cem\u003eAmtrAGL30\u003c/em\u003e and \u003cem\u003eAmtrAGL66/67/104\u003c/em\u003e in genomic blocks collinear with \u003cem\u003eArabidopsis\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e), suggests a strong functional conservation of these genes across plant lineages.\u003c/p\u003e \u003cp\u003eRegarding Type I (Mα) MADS-Box genes, three paralogous genes on chromosome 1 (\u003cem\u003eAmTrH2.01G097100.1, AmTrH2.01G097000.1\u003c/em\u003e: 1.07 and \u003cem\u003eAmTrH2.01G096900.1, AmTrH2.01G097000.1\u003c/em\u003e: 1.08) exhibited Ka/Ks values of \u0026gt;\u0026thinsp;1, indicating positive selection, but no Type II genes had the ratio\u0026thinsp;\u0026gt;\u0026thinsp;1. Type-I MADS-Box genes are known to evolve faster than Type-II genes and experience faster birth and death rates\u003csup\u003e74\u003c/sup\u003e. Unlike Type-II genes, the functions of Type-I genes are still largely unexplored, and they are underrepresented in EST libraries across different plant species\u003csup\u003e14\u003c/sup\u003e. Despite some \u003cem\u003eArabidopsis\u003c/em\u003e Type-I genes being assigned with putative functions, most do not seem to have a functional restraint against non-synonymous mutations, which might explain them having a\u0026thinsp;\u0026gt;\u0026thinsp;1 Ka/Ks value. Selection pressure in MADS-Box genes are generally known to favor purifying selection in other plant species as well\u003csup\u003e75\u0026ndash;78\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGene Structures and Motif Composition of\u003c/b\u003e \u003cb\u003eA. trichopoda\u003c/b\u003e \u003cb\u003eMADS-Box genes/proteins\u003c/b\u003e\u003c/p\u003e \u003cp\u003eOur conserved motif analysis revealed that all MIKC\u003csup\u003eC\u003c/sup\u003e proteins in \u003cem\u003eA. trichopoda\u003c/em\u003e have the typical MIKC domain pattern characteristic of Type-II MADS-Box genes. This included \u003cem\u003eAmtrAGL12\u003c/em\u003e, which is an \u003cem\u003eArabidopsis\u003c/em\u003e ortholog without coiled-coil structure because of lack of some hydrophobic residues\u003csup\u003e13\u003c/sup\u003e. The K-domain, unique to plant Type-II MADS-Box proteins, is not present in other eukaryotes, and likely evolved in the plant Type-II lineage after plant Type-II genes branched off from animal type-II genes\u003csup\u003e13\u003c/sup\u003e. Type-I \u003cem\u003eAmborella\u003c/em\u003e MADS-Box genes have only one or two exons, while Type II genes have more complex structure with six to nine exons. This trend holds true across all plant MADS-Box genes, where Type I MADS-Box genes in plants have a much simpler gene structure with 1\u0026ndash;2 exons than Type II, which typically have 6\u0026ndash;8 exons\u003csup\u003e79\u003c/sup\u003e. The shorter length and simpler structure of Type I MADS-Box genes might have contributed to higher frequency of small-scale duplications than Type-II genes \u003csup\u003e80\u003c/sup\u003e. The diversity of Type II genes in higher plants is probably because of whole-genome duplication events, and retention of the duplicated genes for neofunctionalization, functional subdivision, or balancing the number of genes required for multimerization\u003csup\u003e74\u003c/sup\u003e. The fact that Type-I genes in \u003cem\u003eArabidopsis\u003c/em\u003e are characterized by 1 or 2 exons, and sometimes no intron, and their apparent lack of functionality suggests that Type-I genes are results of reverse transcription and most are pseudogenes without function\u003csup\u003e14\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eExpression of MADS-Box genes in\u003c/b\u003e \u003cb\u003eA. trichopoda\u003c/b\u003e \u003cb\u003efloral buds and flowers\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAnalysis of gene expression in floral buds and mature flowers of \u003cem\u003eAmborella\u003c/em\u003e showed that many MIKC-type genes expressed differentially in males and females. This is in line with the current knowledge that floral transition and patterning genes belong to the Type-II group. The only Type-I genes with differential expression between the two sexes were \u003cem\u003eAmTrH2.04G040100.1\u003c/em\u003e, an Mg gene, which was upregulated in mature male flowers, and \u003cem\u003eAmTrH2.09G063800.1\u003c/em\u003e, an Ma gene, which was upregulated in female floral buds. Expression of Type-I MADS-box genes was not detected in any \u003cem\u003eArabidopsis\u003c/em\u003e tissue examined with microarrays and northern hybridization, which led researchers to conclude that Type-I genes could be non-functional in \u003cem\u003eArabidopsis\u003c/em\u003e\u003csup\u003e81\u003c/sup\u003e. A similar observation of low expression of Type-I genes was reported in another study, leading to an assumption that these genes either have very low expression levels or are expressed under very specific conditons\u003csup\u003e14\u003c/sup\u003e. The first type-I gene to be functionally characterized in \u003cem\u003eArabidopsis\u003c/em\u003e was \u003cem\u003eAGL80\u003c/em\u003e, also known as \u003cem\u003eFEM111\u003c/em\u003e, and it belongs to the Mg clade. \u003cem\u003eagl80\u003c/em\u003e mutants were found to affect female gametophytes after the fusion of polar nuclei with effects on nuclear maturation and vacuole size maintenance\u003csup\u003e82\u003c/sup\u003e. AGL80 was found to interact with the Ma protein AGL61, or DIANA (DIA), in maintaining female gametophyte development in \u003cem\u003eArabidopsis\u003c/em\u003e\u003csup\u003e83\u003c/sup\u003e. In \u003cem\u003eAmborella\u003c/em\u003e, AmTrH2.09G063800.1.p did not phylogenetically nest in the clade with AGL61, but it was related to AmtrAGL62 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), and the gene was upregulated in female floral buds. There is no information in published literature about Ma genes and their expression in floral buds, and this could be a subject of further experimentation. However, several studies have linked Ma genes like \u003cem\u003eAGL61/62\u003c/em\u003e to endosperm, embryo, and female gametophyte development\u003csup\u003e84\u0026ndash;87\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eExpression analysis in floral buds and mature flowers also revealed some differentially expressed genes that were upregulated in different sexes at various stages. \u003cem\u003eAmtrPI-2\u003c/em\u003e and \u003cem\u003eAmtrAP3-2\u003c/em\u003e were upregulated in female buds and male flowers; and \u003cem\u003eAmTrH2.11G126900.1\u003c/em\u003e and \u003cem\u003eAmtrAGL32\u003c/em\u003e that were upregulated in male buds and female flowers. The first two are orthologs of B-function MADS-Box genes, responsible for petal and stamen identity in higher eudicots like \u003cem\u003eArabidopsis\u003c/em\u003e\u003csup\u003e5,8,55\u003c/sup\u003e. AmTrH2.11G126900.1.p nested in the AG/SHP/STK clade in our ML Tree (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and is potentially a C- or D-function ortholog. \u003cem\u003eAmtrAGL32\u003c/em\u003e, on the other hand, is a potential B-sister ortholog. Why B-function genes express differently in these different stages is difficult to explain with available data and literature, warranting further experimentation. Inrestingly, \u003cem\u003eAmTrH2.11G126900.1\u003c/em\u003e showed more than two-fold normalized expression difference between male and female floral buds (log2FC\u0026thinsp;=\u0026thinsp;1.05), and much higher (~\u0026thinsp;16-fold) upregulation in mature female flowers (log2FC\u0026thinsp;=\u0026thinsp;4.09). This upregulation in female flowers could be explained if it were in fact a D-function ortholog specifying ovule identity, but explaining its upregulation in male floral buds remains unclear. Similarly. \u003cem\u003eAmtrAGL32\u003c/em\u003e showed near 21-fold upregulation in male floral buds (log2FC\u0026thinsp;=\u0026thinsp;4.51) and much higher (~\u0026thinsp;154 fold) upregulated in female flowers (log2FC\u0026thinsp;=\u0026thinsp;7.26). In \u003cem\u003eArabidopsis\u003c/em\u003e, \u003cem\u003eAGL32\u003c/em\u003e, also known as \u003cem\u003eABS\u003c/em\u003e (\u003cem\u003eARABIDOPSIS B SISTER\u003c/em\u003e) or \u003cem\u003eTRANSPARENT TESTA 16\u003c/em\u003e (\u003cem\u003eTT16\u003c/em\u003e), is implicated in female-specific pathways such as proanthocyanidin biosynthesis in the seed coat\u003csup\u003e88\u003c/sup\u003e, cell patterning in the sub-epidermal integument cell layer\u003csup\u003e89\u003c/sup\u003e, in the nucellus cell death program\u003csup\u003e90\u003c/sup\u003e, co-ordination of cell division in ovule and seed coat and endosperm formation\u003csup\u003e91\u003c/sup\u003e. Upregulation of \u003cem\u003eAmtrAGL32\u003c/em\u003e in female flowers hints at similar roles of the ortholog in the basal angiosperm. Its upregulation in male floral buds remains unexplained well. \u003cem\u003eAmtrAGL15\u003c/em\u003e was downregulated in female buds too. In \u003cem\u003eArabidopsis\u003c/em\u003e, \u003cem\u003eAGL15\u003c/em\u003e and its paralog \u003cem\u003eAGL18\u003c/em\u003e are implicated in somatic embryogenesis\u003csup\u003e92\u0026ndash;94\u003c/sup\u003e and flowering inhibition\u003csup\u003e95,96\u003c/sup\u003e. Hence, a higher transcript level of the gene in male floral buds could hint at distinct roles in the two sexes, a hypothesis supported by the fact that \u003cem\u003eAmtrAGL15\u003c/em\u003e was downregulated in female flowers. It could also be because of sampling errors if the male buds were collected at a slightly earlier developmental stage than the female buds. However, \u003cem\u003eAGL18\u003c/em\u003e known to be expressed in pollen\u003csup\u003e97\u003c/sup\u003e, could explain \u003cem\u003eAmtrAGL15\u003c/em\u003e\u0026rsquo;s downregulation in female flowers.\u003c/p\u003e \u003cp\u003eWe found no differential expression of \u003cem\u003eAmtrAGL20/42/71/72\u003c/em\u003e between male and female floral buds. \u003cem\u003eAGL20\u003c/em\u003e, known as \u003cem\u003eSuppressor of Overexpression of Constans 1\u003c/em\u003e (\u003cem\u003eSOC1\u003c/em\u003e), is one of the earliest detected MADS-Box genes detected in the apical meristems of mustard after photoperiod-induced flowering\u003csup\u003e98\u003c/sup\u003e. The \u003cem\u003eSOC1\u003c/em\u003e mutants exhibit delayed flowering under both long- and short-day conditions\u003csup\u003e99\u003c/sup\u003e. \u003cem\u003eSOC1\u003c/em\u003e expression can also be induced by vernalization-induced inhibition of \u003cem\u003eAGL25\u003c/em\u003e or \u003cem\u003eFLC\u003c/em\u003e (\u003cem\u003eFLOWERING LOCUS C\u003c/em\u003e), a flowering repressor\u003csup\u003e100\u003c/sup\u003e, and acts as a common point for flowering signals from autonomous, vernalization, and photoperiod pathways\u003csup\u003e101\u003c/sup\u003e. We found no \u003cem\u003eAGL25\u003c/em\u003e (\u003cem\u003eFLC\u003c/em\u003e) ortholog in \u003cem\u003eAmborella\u003c/em\u003e, so it is possible that the floral transition pathway in the basal angiosperm could follow a different pattern than that seen in higher eudicots. This is especially interesting considering how floral organs are spirally arranged in \u003cem\u003eAmborella\u003c/em\u003e, as opposed to whorled in higher eudicots. \u003cem\u003eAmtrAGL22\u003c/em\u003e, an ortholog of \u003cem\u003eAGL22\u003c/em\u003e or \u003cem\u003eShort Vegetative Phase\u003c/em\u003e (\u003cem\u003eSVP\u003c/em\u003e), also, showed no differential expression in floral buds of the two sexes, implying a conserved function of the gene in both sexes\u0026rsquo; floral buds. \u003cem\u003eAGL24\u003c/em\u003e is another gene in the \u003cem\u003eSVP\u003c/em\u003e clade in \u003cem\u003eArabidopsis\u003c/em\u003e, the ortholog of which was not present in \u003cem\u003eAmborella\u003c/em\u003e. \u003cem\u003eSVP\u003c/em\u003e, like \u003cem\u003eAGL24\u003c/em\u003e, regulates \u003cem\u003eSOC1\u003c/em\u003e expression too, but is a flowering repressor unlike \u003cem\u003eAGL24\u003c/em\u003e, despite being phylogenetically related\u003csup\u003e102\u003c/sup\u003e. The absence of an \u003cem\u003eAGL24\u003c/em\u003e ortholog further supports that the mechanism of floral transition in \u003cem\u003eAmborella\u003c/em\u003e could be different from that in higher eudicots.\u003c/p\u003e \u003cp\u003e \u003cem\u003eAmtrAGL7/8/10/70\u003c/em\u003e, the sole gene in the \u003cem\u003eAP1/CAL/SQUA/FUL\u003c/em\u003e subfamily in \u003cem\u003eAmborella\u003c/em\u003e, showed no differential expression between male and female floral tissues. \u003cem\u003eAGL8\u003c/em\u003e has a sequence very similar to \u003cem\u003eAP1\u003c/em\u003e (\u003cem\u003eAGL7\u003c/em\u003e) and \u003cem\u003eCAL\u003c/em\u003e, and it has been found to act redundantly with \u003cem\u003eAP1\u003c/em\u003e and \u003cem\u003eCAL\u003c/em\u003e to promote flower formation in \u003cem\u003eArabidopsis\u003c/em\u003e\u003csup\u003e103\u003c/sup\u003e. \u003cem\u003eAmtrAGL7/8/10/70\u003c/em\u003e did not have differential expression patterns between the two sexes, which hints at a function similar to that of \u003cem\u003eFUL\u003c/em\u003e in \u003cem\u003eArabidopsis\u003c/em\u003e and its involvement in floral transition in both sexes with similar expression levels. In \u003cem\u003eArabidopsis, AGL8\u003c/em\u003e expression is negatively regulated by \u003cem\u003eAP1\u003c/em\u003e expression during the formation of floral meristem and early stages of flower formation/floral organ identity, but \u003cem\u003eAGL8\u003c/em\u003e expression persists in the carpel walls and inflorescence meristems\u003csup\u003e104\u003c/sup\u003e. Since \u003cem\u003eAmborella\u003c/em\u003e lacks distinct \u003cem\u003eAP1\u003c/em\u003e and \u003cem\u003eAGL8\u003c/em\u003e orthologs, it is likely that the regulation of A-function genes in \u003cem\u003eAmborella\u003c/em\u003e differs from that in higher plants. Also, the absence of a distinct \u003cem\u003eAP1\u003c/em\u003e ortholog in \u003cem\u003eAmborella\u003c/em\u003e allows us to make some assumptions about floral primordia formation in the species with spiral floral organs. In higher angiosperms, inflorescence meristems contain spirally arranged primordia, while floral meristem primordia are whorled, corresponding to whorled arrangement of the different floral axes\u003csup\u003e104\u003c/sup\u003e. In absence of more than one gene in the \u003cem\u003eAGL7\u003c/em\u003e family, primordia in the floral meristem might be established spirally, as with inflorescence meristem, hence producing flowers with spirally arranged floral organs. A study showing the presence of \u003cem\u003eAP1\u003c/em\u003e orthologs in basal angiosperms with spirally arranged stamens and carpels, such as \u003cem\u003eMagnolia wufengensis\u003c/em\u003e\u003csup\u003e105\u003c/sup\u003e, conflicts with this hypothesis, however. The study cloned the \u003cem\u003eAP1\u003c/em\u003e ortholog in \u003cem\u003eM. wufengensis\u003c/em\u003e by PCR from a cDNA library, and it was found that \u003cem\u003eMawuAP1\u003c/em\u003e could accelerate flowering and regulate carpel development in \u003cem\u003eMawuAP1-\u003c/em\u003eexpressing wild \u003cem\u003eArabidopsis\u003c/em\u003e but could not recover sepal and petal formation in \u003cem\u003eArabidopsis ap1\u003c/em\u003e mutants. However, the study classified \u003cem\u003eMawuAP1\u003c/em\u003e as a \u003cem\u003eFUL-like\u003c/em\u003e gene (supplementary data\u003csup\u003e105\u003c/sup\u003e). Because \u003cem\u003eMawuAP1\u003c/em\u003e could not recover \u003cem\u003eAP1\u003c/em\u003e function in \u003cem\u003eap1\u003c/em\u003e mutants, and because of high sequence similarity between \u003cem\u003eAP1\u003c/em\u003e and \u003cem\u003eFUL\u003c/em\u003e genes, it is possible that the cloned \u003cem\u003eMawuAP1\u003c/em\u003e gene is more closely related to \u003cem\u003eFUL\u003c/em\u003e than to \u003cem\u003eAP1\u003c/em\u003e, and this can explain spiral arrangement of floral organs in \u003cem\u003eM. wufengensis\u003c/em\u003e as well. Based on these observations, we reaffirm our hypothesis that \u003cem\u003eAP1\u003c/em\u003e orthologs were probably absent in basal angiosperms with spirally arranged floral organs, and \u003cem\u003eFUL-like\u003c/em\u003e genes represent the common ancestral state of the \u003cem\u003eAP1/CAL/FUL\u003c/em\u003e subfamily in angiosperms.\u003c/p\u003e \u003cp\u003eThe differential expression of B-function (\u003cem\u003eAmtrAP3-1, AmtrAP3-2, AmtrPI-1, AmtrPI-2\u003c/em\u003e) orthologs in flowers of the two sexes is our notable finding. Both \u003cem\u003ePI\u003c/em\u003e orthologs are upregulated in males, suggesting a conserved function of \u003cem\u003ePI\u003c/em\u003e genes in the basal angiosperm in stamen identity similar to what is seen in higher eudicots. However, upregulation of \u003cem\u003eAmtrPI-1\u003c/em\u003e in female floral buds suggests that this \u003cem\u003ePI\u003c/em\u003e ortholog might have some \u0026ldquo;rudimentary/atavistic\u0026rdquo; female expression as described by Becker et al. for some B-function genes\u003csup\u003e58\u003c/sup\u003e. This might also be the case with \u003cem\u003eAmtrAP3-1\u003c/em\u003e, where the gene retains an ancestral B-function of being expressed in females. The two genes encode nearly identical proteins but have different lengths because of intronic differences, as described previously. We looked at the \u003cem\u003ecis\u003c/em\u003e-element differences between the two genes and found several elements that were unique to both of them, such as 3-AF1 binding site, ARE, AuxRR-core, LAMP-element, W-box, TCCC-element that were unique to \u003cem\u003eAmtrAP3-1\u003c/em\u003e and ABRE-4, AE-Box, as-1, TGA-element, MYB-like sequence unique to \u003cem\u003eAmtrAP3-2\u003c/em\u003e\u0026rsquo;s promoter regions. The presence of some unique hormone-, light- and stress-responsive \u003cem\u003ecis\u003c/em\u003e-elements in the two genes suggest environmental influences in sex-specific expression of \u003cem\u003eAP3\u003c/em\u003e paralogs in \u003cem\u003eAmborella\u003c/em\u003e, warranting experimental verification.\u003c/p\u003e \u003cp\u003eUpregulation of potential D-function (\u003cem\u003eAmTrH2.11G126900.1\u003c/em\u003e) and E-function (\u003cem\u003eAmtrAGL4\u003c/em\u003e and \u003cem\u003eAmtrAGL 9\u003c/em\u003e) upregulation in female flowers, and no differential expression of the C-function ortholog (\u003cem\u003eAmtrAG\u003c/em\u003e) between the two sexes are indicative of \u003cem\u003eAmborella\u003c/em\u003e plant employing a modification of the fading borders model of floral patterning in basal angiosperms. Kim et al.(2005), studied expression levels of the floral patterning orthologs in different floral organs of basal angiosperms\u003csup\u003e23\u003c/sup\u003e: the expression of \u0026ldquo;\u003cem\u003eAm.tr.PI\u0026rdquo;\u003c/em\u003e and \u0026ldquo;\u003cem\u003eAm.tr.AP3\u0026rdquo;\u003c/em\u003e was high in all floral organs (perianth, stamens, and carpels), and\u0026rsquo;the expression of \u0026ldquo;\u003cem\u003eAm.tr.AG\u003c/em\u003e \u0026ldquo; was the highest in inner stamens and carpels, and \u003cem\u003eAm.tr.AGL2\u003c/em\u003e was expressed in all floral organs of \u003cem\u003eAmborella\u003c/em\u003e, just prior to anthesis. Kim et al. (2004) also cloned two \u0026ldquo;\u003cem\u003eAm.tr.AP3\u0026rdquo;\u003c/em\u003e orthologs and reported the absence of the C-terminal domain in \u0026ldquo;\u003cem\u003eAm.tr.AP3-2\u0026rdquo;\u003c/em\u003e\u003csup\u003e55\u003c/sup\u003e. Our analysis revealed that both \u003cem\u003eAP3\u003c/em\u003e orthologs had complete MIKC domains, likely because we identified the genes using genome data while Kim et al used the transcriptome to characterize the genes. One \u003cem\u003eAP3\u003c/em\u003e ortholog present in the previous scaffold-level assembly of \u003cem\u003eAmborella\u003c/em\u003e, however, lacks a C-terminus as well (data not shown). We find the combination of Kim et al.\u0026rsquo;s RT-PCR and our RNASeq-based approaches useful in enhancing our understanding of floral patterning in \u003cem\u003eAmborella\u003c/em\u003e concerning B-, C-, D-, and E-function genes. Male and female \u003cem\u003eAmborella\u003c/em\u003e flowers show distinct expression patterns of B- and E-function genes, specifically \u003cem\u003eAmtrAP3-2, AmtrPI-1\u003c/em\u003e and \u003cem\u003eAmtrPI-2\u003c/em\u003e are upregulated in male flowers, while \u003cem\u003eAmtrAP3-2\u003c/em\u003e, \u003cem\u003eAmtrAGL4\u003c/em\u003e and \u003cem\u003eAmtrAGL9\u003c/em\u003e are upregulated in female flowers. The genetic consequences of these distinct expression patterns, and if these are associated with sex determination in \u003cem\u003eAmborella\u003c/em\u003e, remain to be investigated. The C-function ortholog \u003cem\u003eAmtrAG\u003c/em\u003e is expressed in the inner reproductive organs in the floral spiral in both sexes, while the D-function ortholog\u0026rsquo;s upregulation in female flowers, suggesting its function in ovule development, as in the classical ABCDE model. Why E-function orthologs are upregulated in female flowers is another question that can be researched further. Based on these observations, we propose that the Fading Borders model of floral patterning in \u003cem\u003eAmborella\u003c/em\u003e employs different B-function orthologs in the two sexes, E-function orthologs are upregulated in females, while C- and D- function orthologs have functions similar to that in the ABCDE model observed in higher eudicots.\u003c/p\u003e \u003cp\u003eRecent research by Carey et al. identified chromosome-9 as the location of sex-determining region (SDR) in the \u003cem\u003eAmborella\u003c/em\u003e\u003csup\u003e106\u003c/sup\u003e. Interestingly, several MADS-Box genes that showed sex-specific up- or down-regulation in \u003cem\u003eAmborella\u003c/em\u003e were found to be located on chromosome-9 in our study as well. Male-biased genes such as \u003cem\u003eAmtrAP3-2\u003c/em\u003e, \u003cem\u003eAmtrAGL20/42/71/72\u003c/em\u003e, and \u003cem\u003eAmreAGL6/13\u003c/em\u003e and female-biased genes \u003cem\u003eAmtrAGL32\u003c/em\u003e and \u003cem\u003eAmTrH2.09G064500.1\u003c/em\u003e were located on chromosome-9, suggesting a strong correlation of expression of MADS-Box genes with sex-determination in \u003cem\u003eAmborella\u003c/em\u003e. The upregulation of the two MIKC*-type genes in \u003cem\u003eAmborella\u003c/em\u003e (\u003cem\u003eAmtrAGL66/67/104\u003c/em\u003e) and (\u003cem\u003eAmtrAGL30\u003c/em\u003e) in male flowers hints at the conserved function of MIKC*-type MADS-Box genes in male gametophyte development. Except \u003cem\u003eAGL67\u003c/em\u003e which is expressed during late embryonic development, the other five MIKC* orthologs in \u003cem\u003eArabidopsis\u003c/em\u003e were found to express exclusively in the pollen, predominantly from the tricellular stage onward, which occurs after the second mitosis\u003csup\u003e107\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo summarize, we identified 42 MADS-Box genes in the \u003cem\u003eAmborella trichopoda\u003c/em\u003e genome, classified and named them with reference to \u003cem\u003eArabidopsis thaliana\u003c/em\u003e orthologs. We could assign sequence-based orthology to 20 of these genes to \u003cem\u003eArabidopsis\u003c/em\u003e MADS-Box genes with Maximum-Likelihood and Reciprocal Best Hit BLAST methods and named them after the assigned orthologies. We conducted structural and functional analyses of the identified genes and based on expression data of floral buds and flowers, we found Type-II MADS-Box genes to be highly expressed, with several genes being differentially expressed between the two sexes, in the bud and mature flower stages, with floral transition-related genes\u0026rsquo; and B- and E-function orthologs\u0026rsquo; expression being highly dependent on the sex of the plant. Our results provide crucial data on updating the fading borders model of floral patterning in basal angiosperms with sex-specific gene expression patterns.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e \u003cb\u003eTaxonomy and Phylogeny of\u003c/b\u003e \u003cb\u003eAmborella trichopoda\u003c/b\u003e \u003cb\u003eMADS-Box Genes\u003c/b\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eWe retrieved the \u003cem\u003eAmborella trichopoda\u003c/em\u003e genome (\u003cem\u003eAmborella trichopoda\u003c/em\u003e var. SantaCruz_75 HAP1 v2.1), coding sequences (CDS), annotation files, and proteome from Phytozome\u003csup\u003e108\u003c/sup\u003e and \u003cem\u003eArabidopsis thaliana\u003c/em\u003e MADS-Box sequences from The Arabidopsis Information Resource (TAIR)\u003csup\u003e46\u003c/sup\u003e. MADS-Box sequences in the \u003cem\u003eA. trichopoda\u003c/em\u003e proteome were retrieved with two rounds of HMMER search with HMMER version 3.3.2\u003csup\u003e42\u003c/sup\u003e. For the first HMMER search, \u003cem\u003eA. thaliana\u003c/em\u003e MADS-Box protein sequences were aligned using Muscle 5.1.linux64\u003csup\u003e109\u003c/sup\u003e accessed from Bioconda\u003csup\u003e110\u003c/sup\u003e, and used the aligned sequences to create a Hidden Markov Model (HMM) profile. This profile was used as the query against the \u003cem\u003eA. trichopoda\u003c/em\u003e proteome with an expect threshold (E) value of 0.1. For the second HMMER search, the MADS-Box HMM profile (PF00319) was downloaded from the EBI-Interpro\u003csup\u003e111\u003c/sup\u003e database, which was used as the query against the \u003cem\u003eA. trichopoda\u003c/em\u003e proteome in a HMMER search with an expect threshold (E) value of 0.1. The sequences were retrieved from the proteome fasta file with a custom-written bash script. We filtered the results from both searches for unique hits, and analyzed the sequences for the presence of the MADS- and K-signature domains against Pfam\u003csup\u003e45\u003c/sup\u003e, Simple Modular Architecture Research Tool (SMART)\u003csup\u003e44\u003c/sup\u003e, and National Center for Biotechnology Information (NCBI) Conserved Domain Database (CDD)\u003csup\u003e43\u003c/sup\u003e databases with InterProScan version 5.52-86.0\u003csup\u003e111\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eFor the phylogenetic analysis, we aligned the full-length MADS-Box protein sequences of \u003cem\u003eA. trichopoda\u003c/em\u003e and \u003cem\u003eA. thaliana\u003c/em\u003e using Muscle, with \u003cem\u003eChara globuralis\u003c/em\u003e MADS Box1 (\u003cem\u003eCgMADS1\u003c/em\u003e)\u003csup\u003e17\u003c/sup\u003e as the outgroup. Phylogenetic analysis was performed using Maximum Likelihood (ML) method in IQTREE2\u003csup\u003e112\u003c/sup\u003e with 1000 bootstraps with the best substitution model. The model (-m) option was set to TEST to choose the best substitution model for tree construction via ModelFinder\u003csup\u003e113\u003c/sup\u003e. The resulting tree file was visualized and annotated in Interactive Tree of Life (iTOL) version 6\u003csup\u003e114\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eHomologies among \u003cem\u003eA. trichopoda\u003c/em\u003e and \u003cem\u003eA. thaliana\u003c/em\u003e MADS-Box proteins were inferred based on nesting of sequences with \u0026gt;\u0026thinsp;50 bootstrap support. For \u003cem\u003eA. trichopoda\u003c/em\u003e scaffolds not resolved with the phylogenetic tree, we used a modification of the reciprocal best hit (RBH) method as employed by Bai et al.\u003csup\u003e78\u003c/sup\u003e. For the RBH method, a protein-protein blast (blastp) was carried out using full length sequences of all \u003cem\u003eA. trichopoda\u003c/em\u003e MADS-box hits as queries against \u003cem\u003eA. thaliana\u003c/em\u003e MADS-Box protein sequences with BLAST\u003csup\u003e115\u003c/sup\u003e. Another blastp was carried out with \u003cem\u003eA. thaliana\u003c/em\u003e MADS-Box protein sequences as query against the \u003cem\u003eA. trichopoda\u003c/em\u003e sequences as the database. Pairs with the best bitscores and E-values from the two BLAST runs were treated as putative orthologs. \u003cem\u003eA. trichopoda\u003c/em\u003e MADS-Box genes were named based on their homologies with \u003cem\u003eA. trichopoda\u003c/em\u003e MADS-Box genes, for instance, \u003cem\u003eAmtrAGLx\u003c/em\u003e for a gene homologous to \u003cem\u003eAGLx\u003c/em\u003e. \u003cem\u003eA. trichopoda\u003c/em\u003e sequences that could not be assigned with orthologous \u003cem\u003eA. thaliana\u003c/em\u003e sequences with both approaches were designated as not orthologous to any \u003cem\u003eA. thaliana\u003c/em\u003e MADS-Box genes and were left unnamed.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\n\u003ch3\u003eAnalysis of Physicochemical Properties and Subcellular Localization\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eWe used the online ExPASy\u003csup\u003e116\u003c/sup\u003e tool to calculate protein lengths, molecular weights, and isoelectric points of \u003cem\u003eA. trichopoda\u003c/em\u003e MADS-Box proteins. Bologna Unified Subcellular Component Annotator (BUSCA)\u003csup\u003e117\u003c/sup\u003e was used to predict subcellular localization of the proteins.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of Conserved Motifs and Gene Structure\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eSequences filtered from the InterProScan search and CDD alignment were subjected to a conserved motifs analysis with Multiple Expectation maximizations for Motif Elicitation (MEME) version 5.4.1\u003csup\u003e118\u003c/sup\u003e with parameters: total number of motifs\u0026thinsp;=\u0026thinsp;10, minimum motif width\u0026thinsp;=\u0026thinsp;6 and maximum motif width\u0026thinsp;=\u0026thinsp;100. We used the subset of the Gene Feature Format (gff3) file of the \u003cem\u003eA. trichopoda\u003c/em\u003e genome to construct the exon-intron map of the \u003cem\u003eA. trichopoda\u003c/em\u003e MADS-Box genes in TBtools-II\u003csup\u003e119\u003c/sup\u003e, accounting for the orientation of the genes. The constructed map was visualized and edited in Inkscape 1.3.2 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://inkscape.org/\u003c/span\u003e\u003cspan address=\"https://inkscape.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eAnalysis of\u003c/b\u003e \u003cb\u003ecis\u003c/b\u003e\u003cb\u003e-regulatory Elements\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe 2000 basepair (bp) upstream regions of the identified MADS-Box genes were extracted from the genome fasta file utilizing the coordinates from the gff3 file with BEDTools\u003csup\u003e120\u003c/sup\u003e, accounting for orientation of the genes. A fasta file prepared with the 2000bp regions was used to identify \u003cem\u003ecis\u003c/em\u003e-elements with PlantCARE\u003csup\u003e121\u003c/sup\u003e. The resulting file was analyzed, and the top 20 most common \u003cem\u003ecis\u003c/em\u003e-elements were visualized in the promoter regions with TBTools-II.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eGene Ontology Analysis\u003c/h3\u003e\n\u003cp\u003eThe fasta files of the identified MADS-Box proteins was subjected to gene ontology analysis with BLAST2GO\u003csup\u003e122\u003c/sup\u003e. The results were visualized with the graphing functionality of MS-Excel.\u003c/p\u003e\n\u003ch3\u003eChromosomal Locations, Collinearity and Evolutionary Selection Pressure Analysis\u003c/h3\u003e\n\u003cp\u003eChromosomal locations of the MADS-Box genes were visualized with a subset of the genomic gff3 file with TBtools-II. Collinearity between the \u003cem\u003eAmborella\u003c/em\u003e and \u003cem\u003eArabidopsis\u003c/em\u003e genomes was examined with the Multiple Collinearity Scan toolkit (MCScanX)\u003csup\u003e123\u003c/sup\u003e with whole-proteomes\u0026rsquo; reciprocal BLASTP results, defining block size as 5, and the resulting collinearity file was used to generate a dual\u003c/p\u003e \u003cp\u003eplot with TBTools-II showing MADS-Box genes in collinear blocks. Ka/Ks ratios among all possible gene combinations were calculated using the Simple KaKs calculator built in TBtools-II.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eGene Expression Data Acquisition and Analysis\u003c/h2\u003e \u003cp\u003eRNASeq fastq files from BioProjects PRJNA748676 (floral buds) and PRJEB38698 (mature flowers) were retrieved from the European Nucleotide Archive (ENA) database\u003csup\u003e124\u003c/sup\u003e. The fastq files were quality-checked with FASTQC\u003csup\u003e125\u003c/sup\u003e and MULTIQC\u003csup\u003e126\u003c/sup\u003e, and trimmed with Btrim\u003csup\u003e127\u003c/sup\u003e as needed. The quality-controlled RNASeq fastq files were aligned against the index file created with \u003cem\u003eA. trichopoda\u003c/em\u003e cds and genome fasta files, and a .txt decoy file created with scaffold ids of the genome with Salmon\u003csup\u003e128\u003c/sup\u003e to create transcript quantification files. The quantification (.sf) files generated from experiments coming from all biological replicates corresponding to a treatment were merged with the quantmerge function in Salmon with the --column argument assigned for numreads and tpm to generate two merged .sf files, which were converted to .csv files. Differential expression analyses were carried out with the DESeq2 package\u003csup\u003e129\u003c/sup\u003e from Bioconductor\u003csup\u003e130\u003c/sup\u003e in RStudio version 2024.4.1.748\u003csup\u003e131,132\u003c/sup\u003e. Expression data for MADS-Box genes from the normalized gene expression data were extracted with a custom R script. log2FC change and p-adjusted (padj) values of genes were plotted in volcano plots with the R package EnhancedVolcano\u003csup\u003e133\u003c/sup\u003e. Differentially expressed genes between treatments were identified based on log2FC values and adjusted p-values (padj). Expression heatmaps were generated using Z-scores of normalized transcripts per million (TPM) values from the raw read counts of MADS-Box genes, and visualized using the pheatmap\u003csup\u003e134\u003c/sup\u003e package from Bioconductor in RStudio. Principal Component Analysis was carried out in RStudio with base R commands, and visualized with the ggplot2 R package\u003csup\u003e135\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":" \u003ch2\u003eConflict of interest\u003c/h2\u003e \u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e \u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis project is supported by the USDA-AFRI (Award # 2022-67037-36254) and South Dakota Agriculture Experiment Station Hatch Project #SD00H800-23 to M.P. Nepal\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eS.P. wrote the codes and scripts, performed the analyses, and wrote the original manuscript. B.A. assisted in writing and reviewing the draft. M.P.N. conceived and supervised the project, frameworked the experiment and analyses, assisted writing of the original manuscript, reviewed, and finalized the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgment\u003c/h2\u003e \u003cp\u003eComputational resources were provided by the High-Performance Computing (HPC) Cluster at South Dakota State University.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe protein and DNA sequences analyzed in this study are accessible in the Amborella trichopoda genome in Phytozome (Phytozome genome ID: 727) (https://phytozome-next.jgi.doe.gov/info/Atrichopodavar_SantaCruz_75HAP1_v2_1). Transcriptomic data of floral buds (BioProject PRJNA748676) and mature flowers (BioProject PRJEB38698) are accessible through the European Nucleotide Archive (https://www.ebi.ac.uk/ena/browser/view/PRJNA748676, https://www.ebi.ac.uk/ena/browser/view/PRJEB38698). Codes used in this study will be provided by the corresponding author, M.P. Nepal ([email protected]), upon request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSimpson, G. G., Gendall, A. R. \u0026amp; Dean, C. When to Switch to Flowering. \u003cem\u003eAnnu Rev Cell Dev Bi\u003c/em\u003e \u003cb\u003e15\u003c/b\u003e, 519-+ (1999). https://doi.org/10.1146/annurev.cellbio.15.1.519\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGonz\u0026aacute;lez-Su\u0026aacute;rez, P., Walker, C. H. \u0026amp; Bennett, T. Bloom and Bust: Understanding the Nature and Regulation of the End of Flowering. \u003cem\u003eCurr Opin Plant Biol\u003c/em\u003e \u003cb\u003e57\u003c/b\u003e, 24\u0026ndash;30 (2020). https://doi.org/10.1016/j.pbi.2020.05.009\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSommer, H. \u003cem\u003eet al. 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R Package Version 1.8. 0 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePackage \u0026lsquo;pheatmap\u0026rsquo; (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eggplot2: Elegant Graphics for Data Analysis by WICKHAM, H (Oxford University Press, 2011).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Amborella trichopoda, Basal Lineage of Angiosperms, MADS-Box Transcription Factors, Fading Borders Model, Flowering Plants","lastPublishedDoi":"10.21203/rs.3.rs-5314709/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5314709/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe ABCDE model is a well-known general model of floral development in angiosperms with perfect flowers, with some modifications in different plant taxa. The Fading Borders Model was proposed to better explain floral patterning in basal angiosperms that typically possess spirally arranged floral organs. The MADS-Box gene family is central to these models and has greatly expanded in higher plants which is associated with increasing complexity in floral structures. \u003cem\u003eAmborella trichopoda\u003c/em\u003e is a basal angiosperm with simpler floral features, and the genetic and functional roles of MADS-box genes in floral development remain poorly understood in the species. The major objectives of this study were to perform a genome-wide identification and characterization of MADS-BOX genes in \u003cem\u003eA. trichopoda\u003c/em\u003e, and to analyze their expression in floral buds and mature flowers t. We identified 42 members of the MADS-Box gene family in \u003cem\u003eA. trichopoda\u003c/em\u003e with a Hidden Markov Model (HMM)-based genome-wide survey. Among them, 27 were classified into Type-II or MIKC group. Based on our classification and orthology analysis, a direct ortholog \u003cem\u003eAPETALA1\u003c/em\u003e (\u003cem\u003eAP1\u003c/em\u003e), an A-class floral MADS-Box gene was absent in \u003cem\u003eA. trichopoda\u003c/em\u003e. Gene expression analysis indicated that MIKC-type genes were differentially expressed between male and female flowers with B-function orthologs: \u003cem\u003eAPETALA3\u003c/em\u003e (\u003cem\u003eAP3\u003c/em\u003e) and \u003cem\u003ePISTILLATA\u003c/em\u003e (\u003cem\u003ePI\u003c/em\u003e) in the species having differential expression between the two sexes, and E-function orthologs being upregulated in female flowers. Based on these findings, we propose a modification in the Fading Borders Model in \u003cem\u003eA. trichopoda\u003c/em\u003e with a modified A-function, B- and E-function orthologs\u0026rsquo; expression being sex-specific, and C- and D-function genes having roles similar to that in the classical ABCDE model. These results provide new insights into the genetics underlying floral patterning in the basal angiosperms.\u003c/p\u003e","manuscriptTitle":"Insights into Genetics of Floral Development in Amborella trichopoda Baill through Genome-wide Survey and Expression Analysis of MADS-Box Transcription Factors","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-28 09:54:16","doi":"10.21203/rs.3.rs-5314709/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-01-13T04:18:19+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-26T11:49:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"214875457158066553700109998539691943991","date":"2024-11-17T23:23:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"68682095194837460629792804068209672687","date":"2024-11-14T06:14:46+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-11-12T06:06:22+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-11-12T05:22:38+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-11-12T01:14:21+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-11-11T05:20:19+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-10-23T00:49:54+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"945ed8a7-9483-42c2-9ba6-53a90f00f827","owner":[],"postedDate":"November 28th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":40776521,"name":"Biological sciences/Evolution/Evolutionary genetics"},{"id":40776522,"name":"Biological sciences/Genetics/Development"},{"id":40776523,"name":"Biological sciences/Genetics/Evolutionary biology"},{"id":40776524,"name":"Biological sciences/Genetics/Gene expression"},{"id":40776525,"name":"Biological sciences/Genetics/Gene regulation"},{"id":40776526,"name":"Biological sciences/Plant sciences/Plant development"},{"id":40776527,"name":"Biological sciences/Plant sciences/Plant evolution"},{"id":40776528,"name":"Biological sciences/Plant sciences/Plant genetics"},{"id":40776529,"name":"Biological sciences/Plant sciences/Plant molecular biology"},{"id":40776530,"name":"Biological sciences/Plant sciences/Plant reproduction"},{"id":40776531,"name":"Biological sciences/Plant sciences/Plant signalling"},{"id":40776532,"name":"Biological sciences/Molecular biology/Transcriptomics"},{"id":40776533,"name":"Biological sciences/Evolution"},{"id":40776534,"name":"Biological sciences/Computational biology and bioinformatics"},{"id":40776535,"name":"Biological sciences/Computational biology and bioinformatics/Classification and taxonomy"},{"id":40776536,"name":"Biological sciences/Computational biology and bioinformatics/Gene ontology"},{"id":40776537,"name":"Biological sciences/Computational biology and bioinformatics/Phylogeny"},{"id":40776538,"name":"Biological sciences/Computational biology and bioinformatics/Protein analysis"},{"id":40776539,"name":"Biological sciences/Computational biology and bioinformatics/Protein function predictions"}],"tags":[],"updatedAt":"2025-02-17T16:02:13+00:00","versionOfRecord":{"articleIdentity":"rs-5314709","link":"https://doi.org/10.1038/s41598-025-88880-x","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-02-12 15:57:35","publishedOnDateReadable":"February 12th, 2025"},"versionCreatedAt":"2024-11-28 09:54:16","video":"","vorDoi":"10.1038/s41598-025-88880-x","vorDoiUrl":"https://doi.org/10.1038/s41598-025-88880-x","workflowStages":[]},"version":"v1","identity":"rs-5314709","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5314709","identity":"rs-5314709","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}