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Structural organization of TRMike genes in representatives of the tribe Phaseoleae DC.
Ekaterina Krylova12, Ksenia Strygina1, and Elena Khlestkina1
1Federal Research Center N. I. Vavilov All-Russian Institute of Plant Genetic Resources, Bol'shaya Morskaya ul., 42-44, Saint Petersburg, 190000, Russian Federation 2Department of Genetics and Biotechnology, Faculty of Biology, Saint Petersburg State University, Universitetskaya nab., 7-9, Saint Petersburg, 199034, Russian Federation
Address correspondence and requests for materials to Ekaterina Krylova, [email protected]
Abstract
Citation: Krylova, E., Strygina, K., and Khlestkina, E. 2021. Structural organization of TFL1-like genes in representatives of the tribe Phaseoleae DC. Bio. Comm. 66(2): 85-108. https://doi.org/10.21638/ spbu03.2021.201
Authors' information: Ekaterina Krylova, Researcher, orcid.org/0000-0002-4917-6862; Ksenia Strygina, PhD, Senior Researcher, orcid.org/0000-0001-6938-1348; Elena Khlestkina, Dr. of Sci. in Biology, Professor RAS, Director, orcid.org/0000-0002-8470-8254
Manuscript Editor: Anton Nizhnikov, Department of Genetics and Biotechnology, Faculty of Biology, Saint Petersburg State University, Saint Petersburg, Russia
Received: August 18, 2020;
Revised: December 29, 2020;
Accepted: January 3, 2021.
Copyright: © 2021 Krylova et al. This is an open-access article distributed under the terms of the License Agreement with Saint Petersburg State University, which permits to the authors unrestricted distribution, and self-archiving free of charge.
Funding: The part of the present work devoted to analysis of VuTFL1.1 and VuTFL1.2 genes was supported by a Saint Petersburg State University grant (Pure ID 62228741). The part of the work devoted to phylogenetic analysis, analysis of nucleotide sequences, predicted elements in promoter regions, predicted amino acid sequences, putative functional domains and 3D protein structures of TFL1, ATC, BFT was prepared within the framework of the state task "Genomic and postgenomic technologies for searching for new genetic markers of economically important traits and new allelic variants of agriculturally valuable genes in the gene pool of cultivated plants and their wild relatives" (VIR project No 0481-2019-0001).
Ethics statement: This paper does not contain any studies involving human participants or animals performed by any of the authors.
Competing interests: The authors have declared that no competing interests exist.
The type of stem growth is one of the key features in determining plant architectonics. Stem growth type is an economically important trait. It interconnects with stem length, flowering duration, yield, resistance to lodging, and suitability of mechanized cultivation. Mutations in the TFL1 gene and its homologs have been demonstrated to change meristem indeterminacy across genera. The aim of this work was to characterize and compare the structural organization of TFL1-like genes in representatives of the tribe Phaseoleae (pigeonpea Cajanus cajan, soybean Glycine max, common bean Phaseolus vulgaris, adzuki bean Vigna angu-laris, mung bean V. radiata, and cowpea V. unguiculata) based on in silico analysis, including analysis of nucleotide sequences, predicted elements in promoter regions, predicted amino acid sequences, putative functional domains and 3D protein structures. We investigated TFL1 (one gene for adzuki bean, four copies for soybean, two copies for other studied species), A7C (two copies for soybean, one gene for other investigated species), and BFT (two copies for soybean, one gene for other studied species) gene family members found in whole-genome sequences databases available for representatives of the tribe Phaseoleae. The presence of duplicated copies for all genes in soybean may be a result of the last genome duplication event during the evolution of this species. Duplication of TFL1 gene to two copies in most of studied species of the tribe Phaseoleae is probably accompanied by the maintenance of the functional state of these genes. The exception is VrTFL1.2 of V. radiata, which likely had lost its functionality. This work broadens the existing data about the number of gene copies, their structural divergence and evolution, and the expected functional differences. This information will be important for understanding the molecular genetic mechanisms underlying the maintenance of indeterminacy in the growth of the shoot apical meristem, as well as in the control of the transition to the reproductive phase of plant development. Keywords: ATC, BFT, Cajanus, Glycine, Phaseoleae, Phaseolus, TFL1, Vigna
Introduction
The transformation of the vegetative shoot apical meristem into the inflorescence meristem and the subsequent formation of the floral meristem and flower organs are important stages in the development of the flower. The initiation of the formation of the floral meristem is well studied in the model plant Arabidopsis thaliana (L.) Heynh. It is controlled by main floral meristem identity genes LFY (LEAFY), AP1 (APETALA1), and TFL1 (TERMINAL FLOWER1) (Benlloch, Berbel, Serrano-Mislata and Madueno, 2007). When LFY expression reaches the critical level, the meristematic function of the apical meristem cells changes, and the floral meristems are laid (Weigel et al., 1992; Weigel and Nilsson, 1995; Bla'zquez, Soowal, Lee and Weigel, 1997). LFY is one of the activators of the AP1 gene, which is also involved in determining the identity of the floral meristem (Benlloch, Berbel, Serrano-Mislata and Madueno, 2007). The antago-
nist of the LFY gene is the TFL1 gene, which maintains the indeterminacy of the activity of the shoot apical meristem, and, thereby, delays the plant transition to flowering (Benl-loch, Berbel, Serrano-Mislata and Madueno, 2007; Moraes, Dornelas and Martinelli, 2019; Périlleux, Bouché, Randoux, and Orman-Ligeza, 2019). TFL1 belongs to the family of phosphatidylethanolamine binding proteins (PEBPs). This family is an evolutionarily conservative group that is found in all taxa from bacteria to animals and plants (Mimida et al., 2001; Benlloch, Berbel, Serrano-Mislata and Madueno, 2007; Jin, Nasim, Susila, and Ahn, 2020). TFL1 belongs to a small gene family CENTRORADIALIS / TERMINAL FLOWER 1 / SELF-PRUNING (CETS). This family in A. thaliana consists of six genes: MOTHER OF FT AND TFL1 (MFT), FLOWERING LOCUS T (FT), TWIN SISTER OF FT (TSF), BROTHER OF FT AND TFL1 (BFT), ARA-BIDOPSIS THALIANA CENTRORADIALIS HOMOLOG (ATC), and TFL1 (Goretti et al., 2020; Jin, Nasim, Susila, and Ahn, 2020). These genes are divided into three groups: (1) the MFT gene that acts during seed germination, (2) a group of FT-like genes (FT and TSF) — floral activators, and (3) a group of TFL1--like genes (TFL1, ATC, BFT) — floral inhibitors (Jin, Nasim, Susila and Ahn, 2020).
FT, TFL1 and TFL1-like proteins interact with FLOWERING LOCUS D (FD) (Benlloch, Berbel, Serra-no-Mislata and Madueno, 2007; Huang, Jane, Chen and Yu, 2012; Ryu et al., 2014). Under inductive conditions FT moves from the leaves through the phloem into the shoot apical meristem and interacts with FD; this complex activates the expression of floral meristem identity genes. Under the non-inductive conditions of a short day, TFL1 forms a heterodimer with the FD protein, which leads to blocking of FD transcriptional activity. Under stressful conditions, BFT competes with FT for binding with FD protein (Huang, Jane, Chen and Yu, 2012). Thus, FT facilitates the plant's transition from vegetative development to the reproductive stage, initiating flowering, and TFL1 acts as a repressor of flowering initiation.
The function of ATC is unclear. Presumably, ATC gene is able to functionally replace the TFL1 in the case of disturbances of its structure (Huang, Jane, Chen and Yu, 2012). However, the phenotypes of atc and tfl1, as well as different levels and patterns of gene expression, suggest that, unlike TFL1, ATC is not involved in determining the identity of floral meristems (Mimida et al., 2001). According to the literature, BFT expression is stimulated only under abiotic stress conditions (Chung et al., 2010; Yoo et al., 2010; Ryu, Park and Seo, 2011; Ryu et al., 2014).
The tribe Phaseoleae DC. includes economically important species, many of which are the most important food legumes in many countries (Boukar et al., 2015). Species of the genus Vigna Savi cowpea (asparagus bean, V. unguiculata (L.) Walp.), mung bean (V. radiata (L.) R. Wilczek.), and adzuki bean (V. angularis (Willd.) Ohwi et Ohashi) are included in the human diet as basic dietetic
products for millions. Many species can provide a wide variety of food products throughout the growing season. According to the Food and Agriculture Organization (FAO), the annual world production of various Vigna species is approaching 12 million hectares in Asia, Africa, Southern Europe, Central and South America. The cowpea VI unguiculata is a multipurpose crop, the interest in which has greatly increased in recent years among breeders (http:// www.fao.org; Boukar et al., 2015; Burlyaeva et al., 2019; Vishnyakova et al., 2019). The mung bean VI radiata has been consumed as a traditional food all over the world for over 3,500 years. Its seeds and sprouts have many compounds important for human health. The adzuki bean is used as food; besides, the mung bean and adzuki bean have long been used in traditional medicine in China.
Genomes of many representatives of the Phaseoleae have been sequenced, but the molecular genetic mechanisms of stem growth control and the role of TFL1 -like genes in this process are not well understood (Schmutz et al., 2010, 2014; Varshney et al., 2012; Kang et al., 2014; Lo-nardi et al., 2019). At the present time information about TFL-like gene copies and their functionality is required for subsequent works. In silico analysis is the basis for future experimental researches of molecular genetic mechanisms in the control of the transition to flowering. The aim of this work was to characterize the structural organization of TFL1 -like genes in representatives of the tribe Phaseoleae.
Materials and methods
The search for homologous sequences of TFL1 (GenBank: AT5G03840) was conducted using the BLASTN algorithm in databases EnsemblPlants (http://plants.ensembl. org/index.html), Phytozome v12.1 (https://phytozome.jgi. doe.gov/pz/portal.html#), Vigna Genome Server (https:// viggs.dna.affrc.go.jp/), and LIS — Legume Information System (https://legumeinfo.org) in the genomes of the main representatives of the tribe Phaseoleae: pigeonpea Cajanus cajan (L.) Millsp., soybean Glycine max (L.) Merr., common bean Phaseolus vulgaris L., adzuki bean VI angu-laris, mung bean VI radiata, and cowpea VI unguiculata (accessed Jan. 20, 2020) (Gonzales et al., 2005; Goodstein et al., 2012; Kersey et al., 2014; Sakai et al., 2015, 2016). The multiple alignment of nucleotide and amino acid sequences was made using MULTALIN v5.4.1 (http:// multalin.toulouse.inra.fr/multalin/) (Corpet, 1988). The cluster analysis was performed using MEGAX software and the Neighbour-joining algorithm with 1000 bootstrap replicates (Felsenstein, 1985; Saitou and Nei, 1987; Tamura, Nei and Kumar, 2004; Kumar et al., 2018). Genes FT (GenBank: AT1G65480), TSF (GenBank AT4G20370), and MFT (GenBank: AT1G18100) were used as the out-group. The prediction of element in promoter regions was carried out with New PLACE (https://www.dna.affrc. go.jp/PLACE/?action=newplace) (Higo, Ugawa, Iwamoto
and Korenaga, 1999) and PlantPAN (http://plantpan.itps. ncku.edu.tw/) (Chow et al., 2019). The annotation of the functional domains was carried out using InterPro (http:// www.ebi.ac.uk/interpro/) (Finn et al., 2016). Modelling of the tertiary structure of the predicted amino acid sequences was made using SWISSMODEL (https://swissmodel. expasy.org/interactive) based on 1wko.1.A, 1qou.1, and 6igi.1 templates from PDB (Waterhouse et al., 2018). For multiple structural alignment of 3D protein structures, the PDBeFold service was used (https://www.ebi.ac.uk/ msd-srv/ssm/). Protein similarity was determined with the LALIGN tool (https://fasta.bioch.virginia.edu/fasta_
www2/fasta_www.cgi?rm=lalign&pgm=lal). The default settings were used for all software.
Results
Identification and phylogenetic analysis of TFL1-like genes of Phaseoleae species
The search for homologous sequences of the TFL1 gene of A. thaliana was carried out in the genomes of the main representatives of the tribe Phaseoleae. In total, four sequences of TFL1 -like genes were identified in the genomes of C. cajan, P. vulgaris, V. radiata, and V. unguicu-
Table 1. Identified homologs of TFL1-\ike genes in the genomes of studied members of the tribe Phaseoleae. * — in concordance with NCBI
Gene of A. thaliana Species Ortholog Sequence Chromosome
TFL1 C. cajan CcTFL1.1 C. cajan10074 3
G. max GmDtl / GmTFLIb / GmTFL1.1.1 AB511820* / Glyma19g194300 19
GmTFL1.1.2 Glyma03g194700 3
P. vulgaris PvTFL1y / PvTFL1.1 JN418231* / Phvul.001G189200 1
V. angularis VaTFL1.1 Vigan04g345000 4
V. radiata VrDetl / VrTFL1.1 Vigrad03g04510 3
V. unguiculata VuTFLI / VuTFL1.1 KJ569523* / Vigun01g173000 1
C. cajan CcTFL1.2 C. cajan36529 Scaffold137665:134,613..135,929
G. max GmTFL1.2.1 Glyma10g071400 10
GmTFL1.2.2 Glyma11g209500 11
P. vulgaris PvTFL1.2 Phvul007g229300 7
V. radiata VrTFL1.2 Vigrad08g05490 8
V. unguiculata VuTFL1.2 Vigun07g059700 7
ATC C. cajan CcATC C. cajan31760 Scaffold132593:212,899..214,119
G. max GmATCI Glyma12g184000 12
GmATC2 Glyma13g317100 13
P. vulgaris PvATC Phvul005g124600 5
V. angularis VaATC Vigan07g072900 7
V. radiata VrATC Vigrad04g03610 4
V. unguiculata VuATC Vigun05g236900 5
BFT C. cajan CcBFT C. cajan16048 8
G. max GmBFTI Glyma09g143500 9
GmBFT2 Glyma16g196300 16
P. vulgaris PvBFT Phvul04g119700 4
V. angularis VaBFT Vigan11g190300 11
V. radiata VrBFT Vigrad01g14660 1
V. unguiculata VuBFT Vigun04g159500 4
Fig. 1. Analysis of phylogenetic similarity of 7R7-like genes (CDS) using Neighbor-Joining method with program MEGAX. Genes FT (GenBank: AT1G65480), TSF(GenBank AT4G20370), MFT(GenBank: AT1G18100) are as outgroup.
lata, three genes in the genome of VI angularis, and eight genes in the genome of G. max. All found sequences are listed in Table 1.
As shown in Figure 1, all identified sequences are divided into three clades. The first clade includes sequences highly homologous to the TFL1 gene of Ara-bidopsis (Fig. 1, yellow). All studied species have two copies of TFL1 in their genome, which we designated as TFL1.1 and TFL1.2. The exceptions are V. angularis and G. max, in the genomes of which there are one and four TFL1 genes, respectively (Table 1).
At the same time, the VaTFL1.1 sequence of VI angularis, according to the Vigna Genome Server database, is located in chromosome 4. However, in accordance with the EnsemblPlants database, this gene is located in chromosome 3, and, according to the LIS database, in chromosome 6. In our study, we present according to the Vigna Genome Server (Table 1).
The other two clades include genes homologous to genes ATC (GenBank: AT2G27550) and BFT (GenBank: AT5G62040) of Arabidopsis (Fig. 1, pink and green clades, respectively). It should be noted that the ATC clade is paraphyletic. In all studied species, except for G. max, one sequence of the ATC and BFT genes was found. In the genome of G. max two copies of these genes were identified (Table 1).
Structural organization of identified genes
The exon-intron structure of most identified TFL1-like genes is the same in all species, with the exception of G. max (see below). Most of the identified genes of G. max, C. cajan, P. vulgaris, as well as VI radiata, VI angularis and V. unguiculata, consist of four exons and three introns (Fig. 2). An exception is the VrATC gene, which consists of two exons and one intron. In addition, the VaATC gene, according to the genomic Vigna Genome Server, consists of four exons and three introns, but according to the EnsemblPlants database, it has five exons and four introns. In our study, we present data in accordance with the Vigna Genome Server.
The second and third exons have highly conserved sequences: in all identified genes the lengths were 62 and 41 bp, respectively (Fig. 2). The first exon was the most variable in length. In contrast to exons, the intron lengths varied greatly in all identified genes; long introns were noted for TFL1 -like genes of C. cajan.
Analysis of the identified G. max genes showed that their exon-intron structures are not the same. Among TFL1 -like genes, according to the genome assembly Glyma.Wm82.a2.v1, GmTFL1.1.2 (Glyma03g194700) consists of five exons and four introns. In the assembly Glyma.Wm82.a1.v1.1, it corresponds to the sequence Glyma03g35250, which consists of four exons and three
introns. In addition, a similar structure was noted for the identified GmATC genes—these genes also have five exons and four introns.
Analysis of promoter regions of TFL1-like genes of V. radiata and V. unguiculata
Due to the fact that the data of TFL1 -like genes of VI an-gularis are contradictory, in further work we will carry out more detailed analysis of TFL1 -like genes of two species of the genus Vigna — V. radiata and V. unguiculata. The search for cis-acting regulatory DNA elements of identified V. radiata and V. unguiculata TFL1 -like genes was in the region ~1000 bp upstream to the ATG start translation codon (Fig. 3, Supplementary file 1). All sequences had strongly conserved motifs, such as CAAT-box and TATA-box. Analyses of promoter elements of all identified sequences revealed many motifs for binding of transcription factors (TFs), light-induced, and hormone-responsible promoter elements. We identified binding sites for proteins that are involved in regulation of genes that are responsive to water stress (Supplementary file 1).
Of all the identified binding sites, the most common were sites for transcription factors containing bHLH domain (basic helix-loop-helix), bZIP TFs, and AP2-like TFs, one is the ERF protein, which regulates plant response to ethylene. In all promoter regions TF binding sites of RAV1 family were identified. Proteins of this family are involved in the regulation of various growth and development processes. In addition, all promoters had multiple binding sites for ARR proteins, which are regulators of gene expression for the primary response to cytokinins. Also, in most promoters (an exception is VuTFL1.1), regions corresponding to the W-box were found. These regions are involved in the response to salicylic acid. Among the identified hormone-sensitive motifs, sites for binding to a cytokinin-binding protein, sites characteristic of gibberellin-regulated genes, as well as sites characteristic of genes regulated by abscisic acid, were found. At the same time, in contrast to the Arabi-dopsis TFL1 gene promoter (Kushwah, Ahmad and Ali, 2014), the analyzed promoter regions did not have binding sites for ARF transcription factors that act as activators or repressors of the transcription of auxin-regulated genes.
Annotation of the functional domains
All predicted proteins belong to the PEPB protein family. Schemes of TFL1-like proteins of V. unguiculata (in comparison with VI radiata) are presented in Supplementary file 2, and all identified domains and critical amino acid residues are designated. Most of the proteins have several overlap domains: PEBP_euk CDD
1500
TFL1.1
1000
ATC
1500
1000
Fig. 2. Length polymorphisms of exons and Introns of Identified 7R7-llke genes. Orange colour — C. cajan
kO
o
TFL1.2
1500
1000
500
exon total total length
BFT
1500
1000
500
lull Dim
e4 exon total total length
A. thaliana, pink — V. angularis, green — V. radiata, blue — V. unguiculata, violet — P. vulgaris, red —
(IPR035810)—specific for PEBP proteins of eukaryotes; domain PEBP (IPR008914)—specific domain for PEBP proteins; and conservative domain IPR001858.
All identified protein sequences have all critical amino acid residues (Asp71, His85, His87, Glu109, Phe120, Asp140) (Fig. 4, Supplementary file 2). Protein VrTFL1.2 has 20 amino acid deletion, but it has all critical amino acid residues. Also, for VrTFL1.2 protein the conservative domain IPR001858 was not found.
In all proteins of V. radiata and V. unguiculata, Phe137 was replaced by Thr137; Ser142 is presented. VuTFL1.1, VuTFL1.2, VrTFL1.1, and VrTFL1.2 contain non-Tyr and non-Trp at position 134 and 138 based on alignment with Arabidopsis FT. These proteins are probably flowering repressors (see discussion).
Presently, ATC is considered the most likely ortho-logue of CEN gene of snapdragon Antirrhinum majus L. (Mimida et al., 2001; Huang, Jane, Chen and Yu, 2012). In our work, we consider ATC in comparison with CEN (Supplementary file 2). All critical amino acid residues (His87-Asp143, Asp73, His89, Glu111, Phe122) are conserved in VuATC.
VrATC protein does not contain PEBP (IPR008914) or conservative (IPR001858) domains. VrATC contains non-tyrosine and non-tryptophan at position 134 and 138 based on alignment with AtFT. In the sequence of the VrATC protein, three important amino acids have been replaced: His86Ile, His88Leu, and Asp72Ser, and so the function of this protein could be lost (Fig. 4, Supplementary file 2).
Analysis of the amino acid sequences of the identified proteins VuBFT and VrBFT showed that the pair of conserved amino acids His88-Asp144 (according to AtTFL1), which determines the specificity of the protein function as a flowering repressor, is preserved. VuBFT and VrBFT contain non-tyrosine and non-trypto-phan at position 134 and 138 based on alignment with AtFT. Thus, these proteins, like TFL1 and ATC, are probably flowering repressors (see discussion).
The prediction of 3D structures of TFL1-like proteins
TFL1. The 1wko.1.A template, deposited in PDB, was used to predict 3D structures of all identified TFL1 proteins. It corresponds to Arabidopsis TFL1 protein with 100 % identity. The applied template achieves about 73.6 % identity with VuTFL1.1, VrTFL1.1 (VrDet1), VuTFL1.2, VrTFL1.2 proteins (Supplementary file 3).
Overall root-mean-square deviation (RMSD) of atomic positions between 3D structures of AtTFL1and VuTFL1.1 is 0.19 A; the same RMSD is found between 3D structures of AtTFL1 and VuTFL1.2 (Supplementary file 4). RMSD between AtTFL1 and VrDet1 is 0.168 A; between VuTFL1.1 and VrDet1—0.122 A. The
smallest RMSD (0.034 A) is between VuTFL1.1 and VuTFL1.2, indicating their high homology of 3D structures. The highest RMSD is between 3D structures of VrTFL1.2 with AtTFL1, VuTFL1.1, VrDet1, VuTFL1.2. This indicates a high degree of divergence of these aligned 3D structures. Sequence identity of protein sequences is the highest (99.4 %) between VuTFL1.1 and VrDet1 (Supplementary file 4). Identity between all TFL1-like proteins of V. unguiculata and VI radiata with AtTFL1 reaches more than 79 %.
ATC. The 1qou.1 template was used to predict 3D structures of all identified ATC-like proteins. It corresponds to snapdragon CEN and it is presented as a di-mer (Banfield and Brady, 2000). It is supposed that protein dimer is unfunctional (Banfield and Brady, 2000).
The applied template 1qou.1 constitutes about 78.61 % identity with Arabidopsis ATC; identity with VuATC and VrATC is 76.61 % and 63.69 %, respectively (Supplementary file 3). The smallest RMSD is between CEN and VuATC (0.187 A) (Supplementary file 4). RMSD between other identified proteins is high, which indicates the low homology of their 3D structures. Sequence identity of protein sequences is about 90 % between VuATC and AtATC, CEN (Supplementary file 4). Identity between VrATC and AtATC, CEN is about 80 %. Amino acid sequence identity of VuATC and VrATC is 86.6 % (Supplementary file 4).
BFT. The 6igi.1 template was used to predict 3D structures of all identified BFT-like proteins. It corresponds to Arabidopsis FT protein. It is presented as monomer. The applied template 6igi. constitutes about 61.11 % identity with Arabidopsis BFT, identity with VuBFT and VrBFT is about 60 % (Supplementary file 3). High RMSD values were obtained by multiple structural alignments of 3D structures of all identified BFT-like proteins. It indicates a high degree of divergence of these aligned 3D structures (Supplementary file 4). Amino acid sequence identity of VuBFT and VrBFT is 98.9 %. Sequence identity of these proteins with AtBFT is about 85 %.
Discussion
The group of TFL1 -like genes includes TFL1, ATC and BFT. There are several studies of the role of the ATC and BFT genes (Mimida et al., 2001; Chung et al., 2010; Yoo et al., 2010; Ryu, Park and Seo, 2011; Huang, Jane, Chen and Yu, 2012; Ryu et al., 2014). It has been shown that the ATC gene is a flowering repressor, which plays a critical role in regulating transition to flowering under non-inductive photoperiodic conditions (Mimida et al., 2001; Huang, Jane, Chen and Yu, 2012). BFT expression is stimulated under certain abiotic stress conditions with a diurnal pattern (Chung et al., 2010; Yoo et al., 2010; Ryu, Park,and Seo, 2011; Ryu et al., 2014).
Fig. 3. The localization of promoter motifs on 5'-upstream region before ATG transcription start site. Orange — light-responsive site, red — TF binding motif, violet — hormone-responsive site.
The TFL1 gene is one of the main regulators of the plant's transition to flowering. Plants with mutations in the TFL1 gene show an early transition to flowering, while the overexpression of this gene showed a prolonged vegetative stage of development, a later transition to flowering with the formation of highly branched inflorescences (Alvarez, Guli, Yu and Smyth, 1992; Ratcliffe et al., 1998; Benlloch, Berbel, Serrano-Mislata and Madueno, 2007). TFL1 homologs have been identified in many angiosperms. The TFL1 genes in soybean G. max and common bean P. vulgaris are the best-studied among the tribe Phaseoleae (Kwak, Velasco and Ge-pts, 2008; Liu et al., 2010; Tian et al., 2010; Kwak, Toro, Debouck and Gepts, 2012; Repinski, Kwak and Gepts, 2012). TFL1 genes were also found in the genomes of the mung bean V radiata, the cowpea V. unguiculata, and also in the genome of the pigeonpea C. cajan (Gumber and Singh, 1997; Dhanasekar and Reddy, 2014; Mir et al., 2014; Li et al., 2018). In the present study, we analyzed the genomes of other members of the tribe Phaseoleae and identified highly homologous genes to TFL1— ATC and BFT.
Identification of new 7FL1-like genes
TFL1. Members of the tribe Phaseoleae are ploidy. Among the considered species, cowpea V unguiculata, mung bean V radiata, adzuki V. angularis, common bean P. vulgaris, pigeonpea C. cajan are diploid (2n=22). G. max is a partially diploidized tetraploid (2n=40), whose chromosomes have undergone many rearrangements (Schmutz et al., 2010).
Four copies of TFL1 in soybean G. max were known. It was shown that these paralogs formed from a whole-genome duplication that occurred about 50 million years ago. Within each pair, genes arose by duplication about 13 million years ago (Tian et al., 2010). In this study, these genes were designated as GmTFL1.1.1 / GmTFL1.1.2 and GmTFL1.2.1 / Gm-TFL1.2.2 (in chromosomes 19, 3, 10 and 11, respectively). Based on our results of the nucleotide sequences analysis, as well as the expression pattern of the GmTFL1.1.1 and Gm-TFL1.1.2 genes (Tian et al., 2010), it was assumed that these genes are maintained in a functional state.
Earlier, three TFL1 genes, PvTFL1x, PvTFL1y and PvTFL1z, were identified in the genome of common
bean P. vulgaris. These genes were mapped in chromosomes 4, 1, and 7, respectively (Kwak, Velasco and Gepts, 2008). In our study, the PvTFL1y gene, which is a functional homolog of the TFL1 of Arabidopsis, was designated as PvTFL1.1 (Fig. 1). Based on the location (chromosome 7) of the PvTFL1.2 identified in the present study, we assume that it corresponds to the previously isolated gene PvTFL1z. Based on our results, PvTFL1x most likely is the PvBFT gene (see below).
In the genome of V. radiata, two TFL1 sequences were previously identified in chromosomes 3 and 8 (Li et al., 2018). It was found that the VrDet1 gene performs functions similar to those of the soybean Dt1 and common bean PvTFL1y (Li et al., 2018). In the present study, this copy was designated as VrTFL1.1. The gene identified in chromosome 8 was designated as VrTFL1.2. Analysis of protein sequence of VrTFL1.2 showed 20 amino acid deletion, and the conservative domain was not identified (Fig. 4, Supplementary file 2). This protein is probably nonfunctional.
In the genome of cowpea V. unguiculata, only one TFL1 gene, VuTFL1, was previously identified and described (Dhanasekar and Reddy, 2014). We identified an additional sequence homologous to the VuTFL1 gene (in the present study designated as VuTFL1.1) in chromosome 7. This copy is designated as VuTFL1.2.
For the first time in the genome of adzuki V. angularis we identified one sequence, VaTFL1.1 (in chromosome 4 according to the Vigna Genome Server), which is highly homologous to the TFL1 gene of Arabidopsis. The genome version (Va3.0) of this species is available on Legume Information System (Kang et al., 2015). Besides that, genome sequencing of adzuki bean using single-molecule real-time (SMRT) sequencing technology is available on Vigna Genome Server (Sakai et al., 2015). According to different databases' information, the chromosomal localization of VaTFL1.1 is different. And we did not identify a sequence homologous to the VaTFL1.1 gene like for other studied members of the tribe Phaseoleae.
Thus, as a result of our study, an additional copy of the TFL1 gene was revealed in the genomes of members of the tribe Phaseoleae (except adzuki) (Fig. 1, clade 1.2). The genes of this group (clade 1.2) are the result of the duplication of genes from clade 1.1 into other chromosomes (Table 1).
ATC u BFT. In the genomes of all the studied members of the tribe Phaseoleae, we identified one ATC and one BFT gene (Fig. 1, Table 1). The exception is soybean G. max, in the genome of which due to the polyploid nature, two copies of ATC were identified in chromosomes 12 and 13— GmATC1 and GmATC2, respectively. In addition, as for other TFL1 -like genes, two homologous BFT genes in chromosomes 9 and 16, GmBFT1 and GmBFT2, were identified in the soybean genome (Fig. 1, Table 1). The genes are likely to be a homoeologous pair resulting from a whole genome duplication.
Based on the location of the ATC and BFT genes, we assume that previously only the BFT gene of the common bean P. vulgaris was identified. This gene was designated as PvTFLlx in the original work and was mapped in chromosome 4 in the P. vulgaris genome (Kwak, Velasco and Gepts, 2008). The activity of this gene in response to stress has not been analyzed. In our study this gene is in the BFT clade (Fig. 1, green clade), and we assume that the PvTFLlx gene is the PvBFT gene.
The results of our study are consistent with the literature data. The identified sequences of highly homologous TFL1 are located in chromosomes 1 and 7 of the V. unguiculata and P. vulgaris (genes VuTFLl.l, VuT-FL1.2, PvTFLl.l and PvTFL1.2). In addition, the VuATC / PvATC and VuBFT / PvBFT gene pairs are located in chromosomes 5 and 4, respectively. The homologous sequences of all genes of the two species V. angularis and VI radiata have been identified in other chromosomes.
Genome synteny of members of tribe Phaseoleae
The genomes of the main members of the tribe Phaseoleae have been sequenced; work on genome sequencing of other species is ongoing (Schmutz et al. 2010, 2014; Varshney et al. 2012; Kang et al., 2014; Lonardi et al., 2019). Due to this, it became known that the genomes are distinguished by a high degree of collinearity (Lonardi et al., 2019). The highest level of macrosynteny was found between the genomes of the cowpea V. unguiculata and the common bean P. vulgaris (Vasconcelos et al., 2015; Munoz-Amatriain et al., 2017). Six of the eleven cowpea chromosomes are largely syntenic with six common bean chromosomes. Each of the remaining five cowpea chromosomes corresponds to parts of two P. vulgaris chromosomes. The exception is chromosomes Vu05 and Vu08 of V. unguiculata (in accordance with the previous numbering, chromosomes 1 and 5, respectively), which have an extended synteny with the P. vulgaris chromosome Pv08 (Lonardi et al., 2019).
Comparative mapping of genomes of three Vigna species (V. unguiculata, V. angularis, V radiata) and common bean P. vulgaris showed that six chromosomes of V. unguiculata (Vu04, Vu06, Vu07, Vu09, Vu10 and Vu11) largely have synteny with single chromosomes of three other species (Lonardi et al., 2019). The presence of rearrangements in chromosomes Vu02, Vu03 and Vu08 suggests that they are characteristic of the divergence of Vigna from Phase-olus, while chromosomal rearrangements in Vu01 and Vu05 are characteristic of the Vigna species.
TFL1-like genes — flowering repressors
Structure of TFL1-like promoters. The plant transition from the vegetative to reproductive phase is controlled by complex factors (Benlloch, Berbel, Serrano-Mislata
and Madueno, 2007). Photoperiod and temperature are the main exogenous factors. In the present study we reveal many light-induced promoter elements in all identified sequences (Fig. 3, Supplementary file 1). Endogenous factors such as phytohormones, circadian clock, and senescence are also important. At the stage of transition to flowering, changes of phytohormonal concentrations occur. In all identified promoter sequences we found many hormone-responsible elements (Fig. 3, Supplementary file 1). It should be noted that the pattern of distribution of identified promoter elements of VrATC was different from other studied sequences.
Structure of TFL1-like and FT proteins. The TFL1 and FT genes encode small proteins with ~60 % identity — 177 and 175 amino acids in length, respectively (Hanzawa, Money and Bradley, 2005; Ahn et al., 2006). A ligand-binding region similar to the structure of mammalian PEBP proteins was previously found in the TFL1 and FT proteins (Banfield and Brady, 2000; Ahn et al., 2006).
In PEBP proteins, the amino acids His86, Asp70, Glu110 and Tyr120 are known to play a critical role in ligand binding. A similar structure was shown for TFL1, FT and CEN plant proteins. Unlike mammalian PEBPs, TFL1 and CEN contain Phe123 and Phe125, respectively, instead of Tyr120, and FT contains Val120 (Ahn et al., 2006).
The amino acids Tyr85 in FT and His88 in TFL1 are located at the beginning of the ligand binding domain and form a hydrogen bond with Glu109 and Glu112, respectively. The study of the proteins' crystal structure, as well as comparison with the CEN protein, have shown that substitutions of His88 in TFL1 and Tyr85 in FT critically affect protein function. Substitutions of His88Tyr in TFL1 and Tyr85His in FT lead to a reversal of the functional significance of proteins — TFL1 becomes an activator of flowering, while the FT protein acquires the function of a repressor (Hanzawa, Money and Bradley,
2005). Earlier, according to the results of experiments with chimeric proteins, it was shown that the replacements in the fourth exon of the TFL1 and FT genes significantly changed the functions of proteins (Ahn et al.,
2006). The sequence was subdivided into four segments (A, B, C, D). It was shown that segments A and C did not differ significantly between TFL1 and FT proteins, while segments B and D were more variable. Segments B and C together are required for FT protein, and segment B or C is required for TFL1. It was shown that the B segment evolves rapidly in TFL1s, but practically does not change in FTs. (Ahn et al., 2006). In our work we will carry out more detailed analysis of TFL1-like proteins of two species, V. radiata and V. unguiculata. In the present study all identified protein sequences (VuTFL1.1, VuTFL1.2, VrTFL1.1, VrTFL1.2) have all critical amino acid residues (Asp71, His85, His87, Glu109, Phe120, Asp140)
(Fig. 4, Supplementary file 2). Besides that, all identified TFL1-like protein sequences of cowpea and mung bean have all four segments (Fig. 4).
Segment B, encoded by the fourth exon, forms an outer loop that determines the fundamental difference in the structure of the FT and TFL1 proteins (Ahn et al., 2006). The loop contains amino acids Gln140 in FT and Asp144 in TFL1. They interact with functionally important amino acids Tyr85/His88. At the same time, only His88 in TFL1 forms a second hydrogen bond with Asp144, while Tyr85 and Gln140 (which corresponds to Asp144) in FT does not form one (Ahn et al., 2006). Our analysis of TFL1-like protein sequences showed that all critical amino acid residues are preserved (Fig. 4, Supplementary file 2).
Further studies of the protein structures of FT and TFL1 identified additional key amino acids for the proper functioning of proteins. It was shown that flowering activators, FT-like proteins, have Tyr134, and flowering repressors, TFL1-like proteins, have an amino acid different from Tyr134. In addition, FT-like proteins have Trp138, and TFL1-like proteins have another amino acid in this position (Ho and Weigel, 2014; Wickland and Hanzawa, 2015). Changes in the amino acid composition in the loop may explain the functional diversification of FT homologues from flowering activators to repressors. Thus, in the beet Beta vulgaris L., two FTs with opposite functions were identified (BvFT1 is a repressor, BvFT2 is a flowering activator). Simultaneous replacement of three amino acids (Tyr134Asn, Gly-137Gln and Trp138Gln) in the loop changes the function of the BvFT1 protein from a flowering repressor to an activator (Pin et al., 2010). In the present study all identified proteins (VuTFL1.1, VuTFL1.2, VrTFL1.1, VrTFL1.2) contain non-tyrosine and non-tryptophan at position 134 and 138 based on alignment with AtFT (Fig. 4).
In addition, the surface charges of FT and TFL1 are crucial for determining the functions. The surface charge of Glu112 in TFL1 is positive, while the charge of Glu109 in FT is negative. The replacement of negatively charged Glu with positively charged Lys (Glu109Lys) in FT leads to a function change, while the replacement of Glu109Asp did not cause the effect (Ho and Weigel, 2014). The replacement of Gln140 with a negatively charged or neutral amino acid (Gln140Asp and Gl-n140Ala) did not lead to a change in the FT function. However, Gln140 replacement with a positively charged amino acid (Gln140Lys and Gln140Arg) leads to the functional change. The charge change within the ligand-binding domain (Asp71Asn) did not affect protein function (Ho and Weigel, 2014).
Thus, the ligand-binding region and the conformation-changing loop are the most important regions for determining the functional significance of proteins, and
1 10 20 30 40 50 GO 70 80 90 100 110 120 13ft WO 150 1G0
I--------+---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+---------4---------+.........+---------+-----
fltTFLl HEHHGTRVIEPLIhGRVVGDVLDFFTPTTKHHVSYHKKQVSHGHELFPSSVSSKPRVEIHGGDLRSFFTLVHIDPDVPGPSDPFLKEHLHHIVTHIPGTTDRTF GKEVVSYELPRPSIGIHRFVFVLFRQ iQRRVIFPNIPSRDH :HTRKFflVE¥DLGLPVflflV
MFLU hnRMPLEPLIVGRVIGEVLDSFTTSTKMTVSYHKKQVYHGHEFFPSSIHIKPKVEIEGGDMRSFFTLIMTDPDVPGPSDPYLREHLHHIVTDIPGTTDRTF GKELVSYEIPKPNIGIHRFVFVLFKQ iRRQCVTPP-SSRDH rHTRNFflnQHELGLPVflflV
VrTFLU HflRMPLEPLIVGRVIGEVLDSFTTTTKhTVTYHKKQVYHGHEFFPSSIHIKPKVEIEGDDhRSFFTLIhTDPDVPGPSDPYLREHLHHIVTDIPGTTDHTF GKELVSYEIPKPHIGIHRFVFVLFKC iRRQCVTPP-TSRDQ NTRSFHHQHELGLPVHHV
VuTFL1.2 MflRVSTDPLVIGRVIGDVLDSFTPTIKHTVTFCKKQVYNGHELFPSTVTTMPRVEIGGGDLRSFFTLIhTDPDVPGPSDPYLREHLHMhVTDIPGTTNflSF GNVLVSYEhPKPNIGIHRFVFVLFQd <RRQCVTPP-SSRDN rNTRKFSSENDLGLPVflflV
MFL1.2 MflRVSTDPLVIGRVIGDVLDSFTPSTKhTVTYSKKQVHNGHELIPST--------------------IhTDPDVPGPSDPYLREHLHMHVTDIPGTTHHSFGHVLVSYEhPKPHIGIHRFVFVLFKQ iRRQCVTPP-SSRDH HTRKFSSEHDLGLPVHRVl
Consensus ,♦.HarvsttPL!iGRV!G4VLDsFTpLtKHbV,KKQV,HGHElfPSb,,♦ ,p( vei.ggd.rsfftl!HtDPDVPGPSDPKLrEHLHHnVTftlPGTTftflsFGnvlVSYEnPkPnlGIHRFVFVLFkQCrRqc!LPp sSRDnpHTRkFss#n#LGLPVRRV%
->
1701 1771
HRQRE TRRRRR NRQRETRRRRR NRQRE TRRRRR NRQRETRRRRR NRQRETRRRrR
B
1 10 20 30 40 50 GO 70 80 90 100 110 120 130 140 150 1G0 170 18182
I--------+---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+-1
CEN HflflKVSSDPLVIGRVIGDVVDHFTSTVKHSVIYNSNNSIKHVYNGHELFPS RtfiTC hRRISSDPLhVGRVIGDVVDNCLQRVKhTVTYNSD—^-KQVYNGHELFPSVVTYKPKVEVHGGDhRSFFTLVMTDPDVPGPSDPYLREHLHHIVTDIPGTTDVSFGKEIIGYEhPRPNIGIHRFVYLLFKQTRRGSVVSVPSY—RDQFNTREFRHENDLGLPVRRVFFNCQRETRRRRR B VuflTC hflHHhISTDPLVIGRVIGDVVDPFTPTVKITVSYNH—KQVYNGHEFFPSSVTTKPKVQIRGGDhRSFFTLLhIDPDVPGPSDPYLREHLHHIVTDIPGTTDTTFGNEVVHYEIPRPHIGIHRFVFFLFKQKCRQflVhKIPSS—RDLFHTRTFREDHDLGLPVflflVFFHRQRETflflRRR VrRTC HNhlSTDPLVIGRVIGDVVDLFSPTVKITVSYNNN—KQVYNGHEFFPSSVRNKPKVQIRGGDrtRSFFTLVPSSFSLPSFITPIFSCISLFHFHflFLSflHDRSRRNEVVSYEIPRPNIGIHRFVFLLFKQKCRQflVhKIPSS—RDLFKTRSFREENDLGLPVflRVFFNRQRETRRRRR Consensus ,,nnn!StDPLv!GRVIGDVVDJ .ptVKitVsYNnn,,♦KqVYNGHEfFPSsVt.kPkVt!rGGDHRSFFTLvn,dpdvPgpsdPylrehlhHivtdipgttD,sfgnE!!sYEiPRPNIGIHRFV^lLFKQkcRqavnkiPss.,RDlFnTR»Fae#N#LGLPVRRVFFNaQRETflRRRR
1 10 20 30 40 50 GO 70
I--------+---------+---------+---------+---------+—
RtFT MSINIRDPLIVSRVVGDVLDPFNRSITLKVTYGQRE-VTNGLDLRPSQVQNKPR RtBFT hSREIEPLIVGRVIGDVLEHFNPSVThRVTFNSNTIVSNGHELRPSLLLSKPR VuBFT HCPSSQSFSLFLVSFQFHSRVHEPLSVGRVIGEVVDIFSPSVRHNVTYSTKE-VRNGHELhPSTVhRKPR VrBFT MPSKSHFSYRLLLKVSLLSHVSFQFMSRLHEPLSVGRVIGEVVDIFSPSVRttNVTYSTKE-VRNGHELhPSTVhRKPR Consensus .♦♦♦♦♦♦♦♦.............„.«sr, JPLiVgRVIGiVltJnpSltUTC.i.eXNGhfLPSx.K
90 100 110 120
170 180
GGEDLRNFYTLVhVDPDVPSPSNPHLREYLHHLVTDIPflTTGTTFlGNEIVCYENPSPTRGIHRVVFILFRQLGRQTVYR-PGHRQNFNTREFflEIYN GGQDLRSFFTLIMMDPDRPSPSNPYhREYLHHhVTDIPGTTDflSF GREIVRYETPKPVHGIHRYVFHLFKCteGRQRVKRflPETREC rNTNRFSSYFG GGDDHRTflYTLIhTDPDHPSPSDPYLREHLHMhVTDIPGTTDVSF GKEIHGYESPKPVIGIHRYVFILFKCIiGRQTVRfl-PSSRDR GGDDhRTflYTLIhTDPDRPSPSDPYLREHLHHhVTDIPGTTDVSFGKDIHflYESPKPVIGIHRYVFILFK iRQTVRH-PSSRDH
.HTL! M.DPDaPSPSttPuiREyLHHtVTDIPgTTd.sF G. #Iv+YE.PkPvaGIHRuVFiLFk(tGRQtV,n+P. .Rtt, :NTr+Fse,+g
GLPVRRVFYNCQRE SGCGGRRL SQPVRRVYFNHQRE TRPRRRP GLPVHHVYFNflQRE TflflRRR GLPVRHVYFNRQRE THRRRR glPVflfiraNaQREtatrrR(t<
-sk->
D
Fig. 4. Multiple amino acids sequences alignment. A — TFL1 -like proteins, B — ATC-like proteins, C — BFT-like proteins. Red is high consensus, blue — low consensus, black — neutral consensus. Yellow designates critical amino acid residues.
GENETICS
Tyr85-Gln140 in FT and His88-Asp144 in TFL1 play a key role in determining the protein function (Hanza-wa, Money and Bradley, 2005; Ahn et al., 2006; Ho and Weigel, 2014). However, despite the fact that in TFL1 of Brassica L. His88 is replaced by Arg (His88Arg), the function of the protein is preserved (Mimida, Sakamoto, Murata and Motoyoshi, 1999). In the present work, all critical amino acids are retained in the identified sequences of TFL1s of V. unguiculata and V. radiata (Fig. 4, Supplementary material 2). According to amino acid sequences these proteins are probably flowering repressors. Based on in silico analysis, including analysis of predicted elements in promoter regions and 3D protein structures, we noticed that the pattern of cis-promoter elements is similar for VuTFL1.1 and VuTFL1.2 (Fig. 3, Supplementary file 1). High homology of 3D structures of these proteins (the smallest RMSD) was identified (Supplementary file 4). Analysis of protein sequence of VrTFL1.2 showed 20 amino acid deletion, and the conservative domain was not identified (Fig. 4, Supplementary file 2). Besides that, the highest RMSD between 3D structures of VrTFL1.2 with other identified TFL1-like proteins (VuTFL1.1, VrDet1, VuTFL1.2) was noticed, and so for VrTFL1.2 a high degree of divergence of other aligned 3D protein structures was detected (Supplementary file 4). On the basis of our results we assume that protein VrTFL1.2 is probably nonfunctional. Additional research is required to clarify this supposition.
In silico analysis of identified protein sequence of VuATC showed that all critical amino acid residues are conserved and this protein contains non-tyrosine and non-tryptophan at position 134 and 138 based on alignment with AtFT (Fig. 4, Supplementary file 2). In the predicted sequence of the VrATC protein, three amino acids that are important for the protein functioning are replaced (His86Ile, His88Leu, Asp72Ser) (Supplementary materials 2). It is known that a charge change within the ligand-binding domain does not affect the function of FT (Ho and Weigel, 2014); therefore, replacing a negatively charged amino acid with an uncharged Ser in VrATC probably does not change its function. However, the 3D structure of VrATC compared to AtATC and VuATC was changed (Supplementary materials 3). Based on in silico analysis, including analysis of predicted elements in promoter regions, predicted amino acid sequences, putative functional domains and protein 3D structures we proposed pseudogenization of VrATC.
The role of BFT is still not fully understood. Based on the results of the first studies, it was suggested that BFT is more similar to FT than to TFL1 (Ahn et al., 2006). A comparison of the amino acid sequences of these proteins showed that in BFT Tyr is located at position 85 (corresponds to Tyr85 in FT). In addition, the sequence of the conserved B segment is more similar between BFT and FT than between BFT and TFL1 (Yoo et al., 2010). The
position homologous to Gln140 / Asp144 (FT / TFL1) in BFT is Glu141 (Ahn et al., 2006). Asp and Glu are negatively charged and have similar biochemical properties.
In the current research, two amino acids that are characteristic of the flowering activator proteins (134Tyr, 138Trp in FT) were not noted in the VuBFT and VrBFT (Supplementary material 2). The conservative amino acid Asn152, characteristic of FT proteins, has been replaced by glycine in BFT. A pair of amino acids critical for functioning as a flowering repressor, His88-Asp144 according to AtTFL1, is preserved in VuBFT and VrBFT.
Based on the obtained data, it can be assumed that most identified proteins could function as blockers of the transition to flowering. Additional studies are needed to establish the possible role of the VrTFL1.2 and VrATC proteins in the transition to the reproductive stage.
Conclusion
Identification and analysis of genes responsible for the type of stem growth are required for the successful selection of modern varieties. The genes characterized in our study belong to the group of TFL1-like genes. In the genomes of all the studied members of the tribe Phase-oleae, we identified duplication of TFL1 gene. V. angula-ris is an exception. The obtained results confirm the high evolutionary conservatism of genes involved in the molecular genetic control of the flowering initiation. Thus, our data based on in silico analysis — including analysis of nucleotide sequences, predicted elements in promoter regions, predicted amino acid sequences, putative functional domains and 3D protein structures — is necessary for the next step of studying the molecular genetic mechanisms underlying the maintenance of indeterminacy in the growth of the shoot apical meristem, as well as in the control of the transition to flowering.
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SUPPLEMENTS
Supplementary file 1
cis-acting regulatory DNA elements identified in TFL1 -like gene promoters of Vigna unguiculata and VI radiata. The localization of promoter motifs on 5'-upstream region before ATG transcription start site. Analysis was performed using New PLACE database. "+" — coding chain, "-" — noncoding chain
Motif Sequence Gene Posistion Description
TFL1.1 67 (-) 347 (+) 654 (-) 956 (+)
TFL1.2 70 (-) 289 (-) 366 (+) 437 (+) 439 (+) 505 (+) 576 (+) 588 (+) 646 (-) 648 (+) 658 (+) 731 (+) 807 (+) 929 (+) 990 (+)
GATABOX GATA ATC 219 (+) 283 (-) 306 (-) 314 (-) 316 (+) 378 (-) 498 (-) 500 (+) 666 (+) 739 (+) 750 (+) 859 (-) 920 (+) S000039
BFT 115 (+) 231 (+) 265 (+) 282 (-) 357 (-) 369 (+) 429 (-) 434 (+) 454 (+) 576 (-) 588 (-) 688 (-) 715 (+) 733 (+) 826 (-) 911 (+)
TFL1.1 179 (-) 180 (-) 317 (-) 347 (+) 360 (+) 489 (+) 559 (-) 578 (+) 606 (-) 696 (-) 697 (-) 874 (+)
GT1CONSENSUS GRWAAW TFL1.2 42 (-) 43 (-) 59 (-) 174 (+) 184 (+) 291 (-) 306 (+) 325 (+) 366 (+) 463 (+) 492 (-) 578 (-) 658 (+) 779 (-) 807 (+) 912 (+) 981 (-) 990 (+) S000198
IA <u ATC 174 (-) 228 (-) 347 (-) 353 (-) 406 (-) 407 (+) 527 (+) 649 (-) 791 (-) 920 (+) 992 (+)
IA OJ #> 'w c o a w <u ■C BFT 56 (-) 208 (-) 231 (+) 284 (-) 396 (+) 429 (+) 443 (-) 508 (-) 735 (-) 828 (-)
IBOX GATAAG TFL1.1 656 (-) S000124
BFT 115 (+) 454 (+)
M TFL1.1 347 (+) 655 (-)
IBOXCORE GATAA TFL1.2 71 (-) 290 (-) 366 (+) 506 (-) 577 (-) 658 (+) 733 (-) 990 (-) S000199
ATC 307 (-) 920 (+)
BFT 115 (+) 231 (+) 283 (-) 429 (+) 454 (+) 734 (-) 827 (-)
IBOXCORENT GATAAGR BFT 454 (+) S000424
TFL1.1 37 (+) 44 (+)
INRNTPSADB YTCANTYY TFL1.2 189 (-) 311 (-) 429 (-) 497 (-) 886 (+) S000395
ATC 350(+) 390 (-)
BFT 9 (+) 446 (+) 550 (+) 847 (-)
SORLREP3AT TGTATATAT BFT 264 (-) S000488
Motif Sequence Gene Posistion Description
ASF1MOTIFCAMV TGACG ATC 777 (+) S000024
TFL1.2 946 (-) 958 (-)
CPBCSPOR TATTAG ATC 617 (-) S000491
BFT 521 (+)
IA <u '¡A ERELEE4 AWTTCAAA ATC 338 (+) S000037
GAREAT TAACAAR TFL1.2 193 (+) 801 (-) S000439
'w c ATC 311 (-)473 (-) 489 (-)
o a <U GARE2OSREP1 TAACGTA BFT 314 (-) S000420
<U c o E TFL1 616 (+) 950 (-)
MYCATRD22 CACATG ATC 662 (+) S000174
.C BFT 422 (-)
PROXBBNNAPA CAAACACC TFL1.2 752 (+) S000263
TFL1.2 670 (-)
WBOXATNPR1 TTGAC ATC 421 (-) 505 (-) 778 (+) S000390
BFT 173 (+) 663 (+) 992 (-)
TFL1.1 169 (-) 219 (+) 320 (+) 443 (-) 622 (+) 862 (-) 892 (-)
TFL1.2 489 (-) 595 (-) 743 (-) 787 (-) 834 (+) 867 (+)
ARR1AT NGATT ATC 8 (-) 141 (+) 259 (+) 331 (-) 409 (+) 540 (+) 572 (+) 582 (+) 621 (-) 793 (+) 837 (+) 848 (+) 936 (-) S000454
BFT 12 (-) 100 (-) 295 (+) 425 (-) 450 (+) 466 (-) 498 (+) 696 (-) 789 (-) 798 (-)
ATHB1ATCONSENSUS CAATWATTG TFL1.2 668 (-) S000317
o E ATHB5ATCORE CAATNATTG TFL1.2 668 (-) S000371
en T3 CRTDREHVCBF2 GTCGAC TFL1.1 296 (-) S000411
!a DPBFCOREDCDC3 ACACNNG TFL1.1 53 (-) 89 (+) 617 (+) 950 (-) S000292
i- HDZIP2ATATHB2 TAATMATTA ATC 932 (-) S000373
BFT 769 (+)
TFL1.1 223 (+) 246 (-)
RAV1AAT CAACA TFL1.2 12 (-) 408 (+) S000314
ATC 66 (+) 577 (-) 703 (+) 825(-) 830 (-) 840 (-)
BFT 252 (+)
Supplementary file 2
o
M
Annotation of functional domains (A) TFLl-like proteins, (B) ATC-like proteins, (C) BFT-like proteins
AtTFLI
20
40
60
74 D t
88H 90H
142S
J12E 123F 137F f 144D
80
100
120
140
160
177 amino acids
PEBP_euk CDD (29-168) PEBP (52-164) conservative domain (68-90)
177
VuTFLI.1
1 20 40
VrDet1/VrTFL1.1
20
40
i
85H
71D , 87H
138S MOD
60
80
100
120
140
85H HO>1T
j"710 | jjai |h°F »j* ^
60
138S 140D i
80
100
120
140
173 amino acids
PEBPeuk CDD (26-164) PEBP (48-162) conservative domain (65-87)
160 173
160 173
173 amino acids
PEBP_euk CDD (33-164) PEBP (48-162) conservative domain (65-87)
VuTFL1.2
1 20 VrTFL1.2
20
85H I^at
r 1 rr'fir
138S 140D,
40
60
80
65H
100
89E _100F
120
118S 120D
40
60
80
100
120
140
173 amino acids
PEBP euk CDD (27-164) PEBP (62-161) conservative domain (65-87)
160 173
153 amino acids
PEBP euk CDD (26-144) PEBP (48-141)
140 153
B
CEN
Er 90H 1QQMI» 1460
181 amino acids
PEBP_euk CDD (27-172) PEBP (54-168) conservative domain (70-92)
20
40
60
80
100
120
140
160
181
AtATC
" 88H 110E 121F 1 * 142D
1 1
175 amino acids PEBP_euk CDD (26-166) PEBP (52-162) conservative domain (66-88)
20
40
60
80
100
120
140
160
175
VuATC
20
40
60
87H
73D f 89H 111E
f ' If ' r f
141S
111E 122F 136Mf 143D
80
100
120
140
160
176 amino acids
PEBP_euk CDD (28-167) PEBP (51-163) conservative domain (67-89)
176
VrATC
20
40
60
« 861
135Mf 140S 110E ^»121F p | 0 142D
80
100
120
140
160
175 amino acids PEBP euk CDD (26-166)
175
GENETICS
o
AtFT
85Y 138W
71D f 87H 109E 120V 134Yf 140Q 152N
I ± ' r f fir^
175 amino acids
PEBP_euk CDD (27-164) PEBP (52-159) conservative domain (65-87)
20
40
60
80
100
120
140
160
175
AtBFT
85Y 139T
71D f 87H 109E 120Y 134K f 141E 153G
r if n r fr—p
20
40
60
80
100
120
140
160
177 amino acids
PEBP_euk CDD (25-165) PEBP (61-149) conservative domain (65-87)
177
VuBFT
t101H
154S
125E -136Y 150Rf 156D ^1680
189 amino acids
PEBP_euk CDD (42-180) PEBP (68-178) conservative domain (81-103)
20
40
60
80
100
120
140
160
189
VrBFT
- 109H 162S
f'Mr I I fir r
176G
197 amino acids
PEBP_euk CDD (50-188) PEBP (76-186) conservative domain (89-111)
20
40
60
80
100
120
140
160
197
Supplementary file 3
(A) Predicted 3D structures of TFL1 gene products of V. unguiculata and V.radiata. lwko.l A template from PDB. (B) Predicted 3D structures of АТС gene products of V. unguiculata and V.radiata. lqou.l template from PDB. (C) Predicted 3D structures of BFT products of V. unguiculata and V.radiata. 6igi.l template from PDB. Green line shows the length of investigating protein sequence as well as blue it's coverage by applied template
AtTFLl
VuTFLl.l
VrDetl / VrTFLl.l
VuTFL1.2
VrTFL1.2
GENETICS
Supplementary file 4
Protein sequence identity and overall root-mean-square deviation (RMSD) TFL1-like proteins, (B) ATC-like proteins, (C) BFT-like proteins
A
AtTFLI VuTFL1.1 VrDetl / VrTFL1.1 VuTFL1.2 VrTFL1.2
AtTFL1 — 92/0.190 91.4/0.168 89.1/0.191 79.9/0.741
VuTFL1.1 92/0.19 — 99.4/0.122 94.8/0.034 86.1/0.716
VrDet1 91.4/0.168 99.4/0.122 — 94.2/0.128 86.1/0.726
VuTFL1.2 89.1/0.191 94.8/0.034 94.2/0.128 — 87.3/0.714
VrTFL1.2 79.9/0.741 86.1/0.716 86.1/0.726 87.3/0.714 —
B
AtATC VuATC VrATC CEN
AtATC — 90.3/0.433 79.9/0.713 96.5/0.408
VuATC 90.3/0.433 — 86.6/0.655 89.8/0.187
VrATC 79.9/0.713 86.6/0.655 — 80.1/0.651
CEN 96.5/0.408 89.8/0.187 80.1/0.651 —
C
AtBFT VuBFT VrBFT FT
AtBFT — 86.8/0.639 87.4/0.953 83.5/0.586
VuBFT 86.8/0.639 — 98.9/0.861 86.6/0.534
VrBFT 87.4/0.953 98.9/0.861 — 87.2/1.013
FT 83.5/0.586 86.6/0.534 87.2/1.013 —
