The importance of plants to humans and the earth is unquestionable. Literature and arts are mostly inspired by the beauty of flowers and plants. Environmental conditions and the plant’s internal factors are two key components that affect plants’ flowering. The number of factors causing flowering may be different but they depend on the plant species, since flower induction can simultaneously be dependent on several factors. Tracking the seasonal changes during the years, type and perception of environmental signals and transducing environmental cues can precede developmental changes of flowering. However, these are not completely understood. The transitions to flowering, many genes have been identified with their pivotal role in the induction and production of floral organs. Postponed flowering in tree species can occur due to their extended juvenility, and this can be a substantial obstacle in breeding programs. The delay in flowering could result not only in a higher amount of biomass but also could occur coincidentally through cross-pollination between genetically modified and native cross-breeding plants. Therefore, it is less likely to have concerns about the movement of transgenes via pollen flow [1]. Two of the most important types of genes that are involved in floral development can be discussed herein. Floral meristem identity genes such as APETALA1 (AP1) and LEAFY (LFY) act as a trigger to push shoot apical meristems (SAM) toward flowering, and floral organ identity genes such as APETALA1 (AP1), APETALA2 (AP2), APETALA3 (AP3), PISTILLATA (PI) and AGAMOUS (AG) have products that are transcriptional factors. The latter is most likely to control the expression of other genes which has products that are involved in the formation and/or function of sepals, petals, stamens and carpels according to the ABC model at the anlagen of SAM [2]. The question concerning the mechanism through which the stimulation of flowering occurs as a result of the long day deserves further investigation. The circadian clock-mediated inductions of CONSTANS (CO) encode zinc finger proteins that regulate the transcription of other genes. This occurs as a result of the long day in Arabidopsis. The sharp increase in CO protein level, as a result of the long day, acts as a transcriptional regulator to stimulate expression in a downstream target gene called FLOWERING LOCUS T (FT) which is a RAF-kinase inhibitor-like protein for accelerating the onset of flowering [3,4]. Genetic screens have yielded the identification of this gene. Both of the aforementioned genes have been identified to be specifically expressed when being in companion with the cells of the leaf. CO–mediated induction of FT can produce a small globular protein (23 kDa) which moves in the phloem from leaves to the SAM [5,6]. FT’s interaction with FD (FLOWERING LOCUS D) up-regulates floral meristem identity genes to induce reproductive development [7-10]. Recent research results indicated that FT can be a candidate in promoting flowering in trees [10]. Meanwhile, the FLC (FLOWERING LOCUS C) is a member of the small family of closely related MADS-domain key protein in plants [11,12] which that acts as a floral repressor by down regulation of the FT gene and mediating autonomous and vernalization pathways. Nonetheless, the FLC has a key role in the initiation of flowering and is involved in the reproductive structure, but its expression during all developmental stages in most parts of the plant propose some new regulatory functions [13]. There are a large number of DNA-binding proteins in plant genomes known as transcription factors (TFs) that bind to the short conserved motifs of 5 to 20 nucleotides called cisacting regulatory elements (CAREs). They orchestrate the initiation of transcription by RNA polymerase II and also which function as gene expression regulators. CAREs are usually found in the vicinity of the 5' end of the genes – usually upstream of the gene transcription start site (TSS), known as a promoter. The identification of plant promoters may provide fundamental information in understanding the regulation of gene expression [14]. Most promoter elements regulating TSS selection are localized in the proximal promoter. Many plant promoter databases have been developed based on cis-regulatory elements including PlantCARE [15], http://www.bioinformatics.psb.ugent.be/webtools/plantcare/html/], PLACE [16], http://www.dna.affrc.go.jp/PLACE/] or TRANSFAC [17], http://www.gene-regulation.com/pub/databases.html] and ppdb [18], http://www.ppdb.gene.nagoya-u.ac.jp] and PlantPan [19], http://plantpan2.itps.ncku.edu.tw.

The identification of direct physical binding of proteins or indirect protein-protein interactions could give some insight into the discovery of new molecular players. It further promotes or strengthens the interpretation of screens relating to genome-wide association, thereby giving clues about the biological function of some protein and other properties observed thereof.

Previously, researchers have used the STRING database [20] to find direct and indirect interactions of proteins with FT and FLC. The objectives of the present work were to recognize the cis-elements modules and their organization in the regulatory promoter rejoin of FT and FLC genes in Arabidopsis and peach. This was done in order to generate a comprehensive understanding of the regulation of gene expression. Furthermore, this research endeavored to identify the common motifs between FT and FLC proteins. Their functions through different data banks were studied and, finally, the proteins that interacted with FT and FLC were recognized. The proteins were hypothesized to be involved in the acceleration or suppression of flowering.

Promoter analysis of FT and FLC genes

The genomic DNA of FT and FLC gene in Arabidopsis (NM_105222, NM_001085094, respectively) was accessed through the National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/) web server. It was applied as a platform in order to recognize the promoter region of the FT and FLC genes by using the BLAST search through the Phytozome database (http://www.phytozome.net/). These were used for the purposes of investigation in this study. After identifying the genes on the chromosome using the BLAST-N algorithm, the region around the 1500 bp upstream of the start codon (ATG) in FT and FLC genes of P. persica and Arabidopsis were taken as a promoter. The upstream region of the FT and FLC in Arabidopsis and their corresponding orthologs in P. persica were analyzed by using the PLANT CARE and PlantPan database. For this purpose, upstream region sequences of FT and FLC in Arabidopsis and their corresponding orthologs were applied to predict their key cis-acting regulatory elements and the precise location of these elements.

Motifs identification and their functional analysis

Referring to the FLC and FT proteins obtained from the NCBI, these sequences were aligned by the MEME web server (http://meme.sdsc.edu/meme4_6_1/cgi-bin/meme.cgi) so as to identify their common motifs [21]. In the case of FT, 91 sequences with an average length of 176.6 amino acids were analyzed. In FLC, however, 58 sequences with an average length of 184.6 amino acids were analyzed. In both proteins, the minimum and maximum motif size was 6 aa and 50 aa, respectively. The default value of 15 and 10 has been determined empirically for the maximum motif number concerning the FLC and FT, respectively. The predictions of probable functions of conserved domains of FT and FLC within the protein sequences was performed by the ELM program (http://elm.eu.org) and SMART (http://smart.embl-heidelberg.de). The UniProtKB (http://www.uniprot.org/) was used for the identification of some gene ontology characteristics of FT and FLC. Primary sequence analysis was done by ProtParam (http://expasy.org/cgi-bin/protparam). Moreover, similarity was assessed by using different programs at NCBI, such as the BLAST-P and PSI-BLAST (http://BLAST.ncbi.nlm.nih.gov/BLAST.cgi). Multiple sequence alignments were performed by using the Vector NTI Suit 9.

Secondary and tertiary structure prediction

The Swiss model web server (http://swissmodel.expasy.org/) was employed for the prediction of secondary structure of FT and FLC in A. thaliana.

Protein-protein interaction networks

A well-defined protein–protein interaction network in Arabidopsis gives good reason for the use of FLC (NP_001078563.1) and FT protein (NP_176726.1) as a query. STRING 9.0 (http://string-db.org) was used to predict all the proteins that interact with the FT and FLC proteins.

Analysis of FT and FLC promoters

The results of FT and FLC promoter analysis on the 1.5 kb sequence upstream of the ATG (start codon) in Arabidopsis and their orthologs in P. persica by PLANT CARE revealed that different transcription factors (TF) are attached to specific DNA binding sites. The TF mediate FT and FLC gene expression (Figure 1) in which there are the light responsive elements (LRE), hormone-responsive element (HRE), stress-responsive element (SRE) and some miscellaneous-responsive elements (MRE). Each of these elements has its own specific cis-acting regulatory element.

Figure 1: FT promoter sequence of Arabidopsis thaliana. 1500 bp upstream of start codon were used to represent proximal transcriptional DNA binding site (TDBS). Grey section represent CAAT and TATA box core promoter region. Red cis-regulatory elements involved in light responsive element. The colorless box indicated cis-regulatory element involved in other responses. The functions of these specific arenas are addressed in Table 1.

For example, the subdivisions of HREs include the abscisic acid-responsive element, gibberellins-responsive element, auxin-responsive element, ethylene-responsive elements and the jasmonic acid and salicylic acid-responsive elements (Table 1). This indicates that the responses to photoperiodic and circadian rhythms closely correspond with TFs that affect the stress- and hormone-responsive elements. It may suggest that the integration of TFs involved in hormone and stress can control FT and FLC expression which lead to modulating light responses [22]. This hypothesis has to be experimentally supported by a HRE defected mutant. Results originating from bioinformatics results about cis-acting regulatory elements on FT and FLC promoters that are involved in endosperm and meristem expression are consistent with previous reports [13,23]. The specific elements presented in the FT and FLC promoters of Arabidopsis and peach were counted and are depicted in Tables 1 and 2. It has been stated that the PLANT CARE promoter database focuses on cis-regulatory elements rather than the core promoter structure. This approach will lead to a better understanding of the gene expression profile [18]. The core promoter is comprised of the least sequence required for gene expression which usually occupies around 80 nucleotides surrounding the transcription start site [2]. The particular time and place for expressive activity are designated by the transcription factor binding site (TFBs). It is known that the TFBs are located in the non-coding sequence upstream of the transcription start site called the regulatory promoter region. Accordingly, recognizing the mentioned cis-regulatory modules and their organization is a manifest need to shed light on the regulation of gene expression [24].

Table 1: Comparison between FT cis-regulatory elements of Arabidopsis and peach resulted from 1500 bp upstream of the ATG.

Table 2: Comparison between FLC cis-regulatory elements of Arabidopsis and peach resulted from 1500 bp upstream of the ATG.

Results indicated that the maximum number of elements in both plants is not allocated to LRE according to PLAN CARE. This magnifies the role of other cis-elements during the flowering process. Among the LRE elements in both genes in the two species of Arabidopsis and P. persica, the G-box had the highest cis-acting regulatory element involved in light responsiveness. G-box is not only a target site of phytochrome interacting factors (PIFs), which is required for phytochrome-regulated transcription in photoperiod response [25], but it is also involved in stress and defense responses [13,26]. Of the total number of responsive elements identified in FT and FLC, Arabidopsis had the highest number. This may not be due to the amino acid length of FT and FLC in Arabidopsis (Table 3). Perhaps it is due to the day neutral characteristic of P. persica in contrast to that of Arabidopsis which is a long-day species.

Table 3: Evaluation of the FT and FLC amino acids length between Arabidopsis thaliana and other species.

The research results by the PlantPan database indicate that there are several common TFBS in FT and FLC promoters which were not recognizable by PLAN CARE. Some of the specified TF in regulatory promoter regions of the FT and FLC gene were AG [27], LFY [28] and AP1 [29]. FLC and its target (FT) contained a CArG box [30] in their binding regions. Too many MYB and MYC TF were found also at FT and FLC promoters by the PlantPan [31,32]. AG and AGLs are TFs that work with SOC1 in flowering. In the presence of external or internal stimuli, they may motivate floral meristem identity genes like LFY and AP1. This often results in flower development at the anlagen of SAM [2].

Motifs identification

Flowering locus T is a probable component of the mobile flower promoting signal (floral stimulus or florigen). It promotes the transition from vegetative growth to flowering. Its subcellular location is the cytoplasm and the nucleus. In contrast to FLC that is mostly localized in shoot apexes (add references or reason); the FT is mostly localized in the vasculature of leaves. The FT protein product moves from leaf to shoot apex and acts as a long-distance signal that induces Arabidopsis flowering [9,33]. The MEME algorithms that have been widely used for the discovery of DNA and protein sequence motifs [34] were used to identify conserved motifs of the FT proteins deposited in the NCBI data base. It is clear from Figure 2 that three domains are devoted largely to the total protein. The evaluation of the aforementioned domain function by using the SMART web server revealed that the first motif is obviously a PEBP (Phosphatidyl ethanolamine-binding protein) family – a highly conserved group of proteins that have been identified in numerous tissues in a wide variety of organisms, including bacteria, yeast, nematodes, plants, drosophila and mammals [3]. Various functions have been described for the members of this family, including the control of the morphological switch between the shoot growth and flower structures as well as the regulation of several signaling pathways such as the MAP kinase pathway [35]. The domains that were mentioned above can be evaluated by using the eukaryotic linear motif resource for functional sites in proteins (ELM) that have been revealed following the functional site class: Motif 1: APCC-binding Destruction motifs act by the anaphase-promoting ubiquitin ligase complex APC/C and selectively target numerous cell cycle-regulatory proteins for ubiquitin-mediated proteasome-dependent degradation, BRCT phosphopeptide ligands (or the BRCT domains in Eukaryotes which are present in proteins that are associated with the DNA damage-response. They recognize and bind specific phosphorylated serine (pS) sequences. This phospho-protein mediated interaction of the BRCT domain has a central role in the check points of the cellcycle. Motif 2: the PKA Phosphorylation site (of motifs phosphorylated by a subset of AGC group kinases including PKA that all have similar sequence specificity). The WXXXYF motif is repeated in the Pex5p protein and is bound non-conventionally by an SH3 domain in the Pex13p peroxisomal membrane protein. It is involved in the import of peroxisomal matrix proteins. Motif 3: AP2 alpha ligands (concerning motifs responsible for the binding of accessory endocytic proteins to the alpha-subunit of adaptor protein AP-2 and their recruitment to the site of clathrin coated vesicle formation). This protein contains two copies of an approximately 70 amino acid domain termed the AP2 repeat because of its initial description in the floral homeotic protein APETALA2 (AP2) [36]. Evidence shows the connection of the AP2- domain in both ethylene and JA signaling. This suggests that ethylene and JA may cross-talk via these transcription factors [37].

Figure 2: Distribution of FT protein motifs using MEME web server.

The PIKK phosphorylation site often known as the phosphoinositide-3-OH-kinase related kinases (PIKKs) are atypical protein kinases exclusive to eukaryotes. The PIKK members are large proteins with Ser/Thr kinase activity serving important roles in DNA repair and DNA damage checkpoints, and also in the PKA Phosphorylation site (concerning motifs phosphorylated by a subset of AGC group kinases including PKA, all of which have similar sequence specificity).

Flowering locus C (FLC) is a MADS-box eukaryotic family of transcriptional regulators that share a stereotypical MIKC structure [38]. They have a central role in the regulation of flowering time in late-flowering phenotypes. This can enable the blocking of the transition from the vegetative to the reproductive development by repressing the 'SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1' and FT gene [39]. Results of the electronic Fluorescent Pictograph (eFP) Browser showed a high FLC expression in the vegetative shoot apex and also in the root tissues. A lower level of expression was observed in leaves and stems (rephrase this). Its subcellular localization is probably the nucleolus based on the eFP Browser.

It appears that in plants, FLC has more frequent domains compared to FT (Figure 3 and Table 4). According to the ASMART web server, FLC is a MADS-box domain that seems to be more closely related to SRF (Human serum response factor) domains which is a ubiquitous nuclear protein important for cell proliferation and differentiation as well as MEF2 domains in fungi. Alvarez-Buylla et al. [38] have suggested that animal and fungal MEF2-like sequences are more closely related to the t plant MADS-domain sequences than that of animal SRF like sequences. Proteins belonging to the MADS family function as dimmers. MADS genes in plants encode key developmental regulators of flower, fruit, leaf and root development. Eukaryotic regulatory proteins with the highly conserved DNA-binding MADS domain include the MCM1 which is the regulator of cell type-specific genes in fission yeast, the DSRF which is a Drosophila trachea development factor, the MEF2 family of myocyte-specific enhancer factors and the Agamous and Deficiens families of plant homeotic proteins. According to the ELM server, the functional site classes were obtained with the FLC motif, including the Y-based sorting signal which is responsible for the interaction with the mu subunit of AP (Adaptor Protein) complex, the PKB Phosphorylation site which hosts a AGC group of kinases that act as a phosphorylation factor, the Cyclin recognition site, the PP1 docking motif, the SID, the PLK phosphorylation site, the Clathrin box, the MAPK docking motif, NLS classical Nuclear Localization Signals, di Lysine ER retrieving signal, FHA phosphopeptide ligands and the PIKK phosphorylation site. Consensus sequences in each motif of the FT and FLC, in addition to the amino acid frequency in FT and FLC, are presented in Tables 5 and 6.

Figure 3: Distribution of FLC protein motifs using web server of MEME.

Table 4: FT and FLC amino acid length comparison, originated from NCBI.

Table 5: Motif distribution.

Table 6: Frequency of FT and FLC amino acids in plant exist in NCBI data bank.

Analysis of protein-protein interaction network

GIGANTEA (GI) is presented as a FB in Figure 4. It has a protein protein interaction with CO and FT, but acts earlier than CO and FT in a circadian clock-controlled flowering pathway. This is because the GI is mediated in the regulation of phytochrome B signaling along with the CO to promote the FT gene when exposed to the photoperiod of long-day [40]. In addition to GI, CO can be influenced by two other transcription factors including HRB1 and SPINDLY is a zinc finger domain that could be involved in red and blue light signal transduction. Meanwhile, (SPY) acts as a repressor of GA responses. Furthermore, the positive regulation of cytokinin signaling can have an indirect and direct interaction with CO and FT, respectively [41,42].

Figure 4: Protein-protein interaction network analysis of FLC (left) and FT (right) using STRING 9.0 in Arabidopsis thaliana.

Regarding the FLC protein, as is clear from Figure 4, the FLA represents FRI (FRIGIDA) and is a dominant allele that directly interacts with FLC to keep the plant in its vegetative state. According to the STRING web server, EMBRYONIC FLOWER 2 (EMF2) is shown as CYR1, CURLY LEAF (CLF) and FERTILIZATION-INDEPENDENT ENDOSPERM (FIE). It is similar to the polycomb group protein that may be involved in flowering processes by repressing FLC promoters. The FLOWERING LOCUS D (FLD) is similar to the aforementioned proteins as it suppresses the FLC function. FLC can also interact directly with the SHORT VEGETATIVE PHASE (SVP) by binding to its promoter region to delay flowering (references). However, it has been stated that when the SVP loses its function, it does not fully suppress the delay of flowering because, according to Figure 4, there are some other proteins with which the FLC may interact [43]. Another key TF that interacted with FLC is VRN2 (Figure 4). It has been reported that the activation of VRN genes, after a long period of cold, can encode a DNA-binding protein and, especially, a homologue of one of the polycomb group proteins. The encoding is skewed towards the downregulation of FLC by dimethylation of lysines 9 and 27 on histone [44]. According to string results, the FRIGIDA-LIKE1 (FRL1) can directly or indirectly up-regulate FLC proteins [45].

The research results gained by protein-protein interaction indicated that the domains involved in the developmental and reproductive pathways throughout the plant life are closely connected with the other proteins that act as a switch from the vegetative stage to the reproductive development like the bZIP transcription factor FD, AP2, SVP and so forth. The research results of secondary structure predictions are purely indicative of a high rate of helix in FLC protein structures than the FT (Figure 5). Frequency of versatile regulatory elements on FT and FLC promoters suggest that these genes recruit several TFs to regulate specific gene expression. This issue is evident in their protein-protein interaction with STRING web server’s results. The AP2-motif that was revealed in motif analysis of the FT protein is shown to be involved in stress and hormone-responsive gene expression [37] which, to some extent, explains the presence of hormonal and stress responsive elements on the regulatory promoter region of FT gene. The AP2 domain can be shown to correspond with ethylene and the MeJA responsive element, as also reported previously [37].

Figure 5: Secondary structure prediction of FT (left) and (FLC) in Arabidopsis thaliana using http://swissmodel.expasy.org/.

Furthermore, the analysis of genes involved in the flowering process confirmed that the response to photoperiodic and circadian rhythms may closely correspond with TFs that affect stresses and hormone responsive elements. Accordingly, it can be reasonably concluded that manipulating the plant flowering process precisely, without affecting the other normal physiological processes, can be a difficult ambition to achieve.

Author declares no competing financial interest.