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Comprehensive Evolutionary and Expression Analysis of FCS-Like Zinc finger Gene Family Yields Insights into Their Origin, Expansion and Divergence

Plant evolution is characterized by frequent genome duplication events. Expansion of habitat resulted in the origin of many novel genes and genome duplication events which in turn resulted in the expansion of many regulatory gene families. The plant-specific FCS-Like Zinc finger (FLZ) gene family is characterized by the presence of a FCS-Like Zinc finger (FLZ) domain which mediates the protein-protein interaction. In this study, we identified that the expansion of FLZ gene family size in different species is correlated with ancestral and lineage-specific whole genome duplication events. The subsequent gene loss found to have a greater role in determining the size of this gene family in many species. However, genomic block duplications played the significant role in the expansion of FLZ gene family in some species. Comparison of Arabidopsis thaliana and Oryza sativa FLZ gene family revealed monocot and dicot specific evolutionary trends. The FLZ genes were found to be under high purifying selection. The spatiotemporal expression analyses of Arabidopsis thaliana FLZ gene family revealed that majority of the members are highly expressed in reproductive organs. FLZ genes were also found to be highly expressed during vegetative-to-reproductive phase transition which is correlated with the proposed role of this gene family in sugar signaling. The comparison of sequence, structural and expression features of duplicated genes identified lineage-specific redundancy and divergence. This extensive evolutionary analysis and expression analysis of Arabidopsis thaliana FLZ genes will pave the way for further functional analysis of FLZ genes.


Introduction
Genome duplication and accumulation of variation are prerequisites to the evolution of biological complexity. The origin and diversification of embryophytes are accomplished due to massive changes in the genomes. Whole genome duplications (WGD) and subsequent rearrangements such as gene loss shaped most of the plant genomes [1,2]. Paleopolyploidy is a recurrent feature in the evolution of land plants [3][4][5][6]. Comprehensive phylogenomic analyses revealed that evolution and diversification of seed plants happened due to two ancient WGD events; one predates the divergence of gymnosperms and angiosperms and the other predates the diversification of angiosperms [7]. Similarly, many lineage and species-specific WGD events are revealed in the analysis of many monocot and dicot genomes [1][2][3][4][8][9][10][11][12][13].
A high percentage of plant genes are grouped into gene families based on sequence similarity features such as the presence of a common protein domain. The expansion and contraction of specific gene families are correlated with the characteristics of the specific species [6,11,12,[14][15][16][17]. Analysis of gene family evolution in plants and metazoa identified that gene families undergo species-specific contraction and expansion [18,19]. The expansion of gene numbers due to duplication events and subsequent divergence are major contributors to the evolution of biological complexity [20].
The FCS-Like Zinc-finger (FLZ) gene family is a plant-specific gene family characterized by the presence of a FCS-Like Zinc-finger (FLZ) domain [21]. FLZ domain possesses a typical alpha-beta-alpha topology and mediates the protein-protein interaction. [21,22]. Functional analysis revealed that many members of this gene family is related to plant development, senescence, stress mitigation and sugar signaling [22][23][24][25][26][27]. Although, the role of many FLZ genes in the regulation of plant life is known, not much information is available on the evolutionary aspects of this gene family.
A comprehensive survey of FLZ domain containing proteins from sequenced plant genomes was done in our earlier study [21]. The availability of Amborella trichopoda genome, which is a basal angiosperm with no lineage-specific WGD event, enables detailed studies on the evolutionary expansion of gene families in higher angiosperms [3]. In this study, using all these available resources, we tried to construct the evolutionary history of FLZ gene family. We analysed the evolutionary expansion of FLZ gene family and correlated it with already established genome duplication events. Detailed phylogenetic studies of dicot and monocot models were done and the evolutionary features were compared. The selection pressure on FLZ genes was studied within and across the genome. The spatiotemporal expression dynamics of Arabidopsis thaliana FLZ gene family was studied and expression divergence of duplicated gene members was analysed.

Phylogenetic analysis
In order to analyse the evolutionary relationship among FLZ genes of different species, fulllength proteins were used for phylogram construction. The sequences were aligned by ClustaX 2.0 [32]. Both Bayesian inference (BI) and maximum likelihood (ML) methods were used for phylogenetic analysis. MrBayes(v. 3.2.5) was used for BI analysis and MEGA (v. 5.0) was used for ML analysis [34,35]. ProtTest 2.4 was used for identification of the best suited model of amino acid substitution [36]. Trees generated using BI method used JTT model of amino acid substitution [37]. In all BI analysis, two Metropolis-Coupled Markov Chain Monte Carlo runs each with 16 chains was performed with default settings for 5,000,000 generations. Approximately 200,000 trees were generated during the process and 25% of the generated trees were disregarded as burn-in. The BI trees were edited and visualized using FigTree (v. 1.4.2) [38].
ML trees were constructed with JTT model of amino acid substitution with 1000 replicate bootstrap analysis. The FLZ proteins from different species were named according to their relationship with A. thaliana FLZ1 protein (S1 Table). The duplication data of FLZ gene family was retrieved from PLAZA v. 2.5 (http://bioinformatics.psb.ugent.be/plaza/versions/plaza_v2_ 5/) which is based on i-ADHoRe program which detects homologous genomic regions [39,40]. The duplication data was manually curated and verified by analysing the relative position of genes annotated as duplicates by the program in the phylogram of individual species (Data not shown). The chromosomal location of FLZ genes were determined by Whole Genome Mapping tool available in PLAZA v. 2.5 (http://bioinformatics.psb.ugent.be/plaza/versions/ plaza_v2_5/genome_mapping/index) [39]. The gene and protein structure was constructed using GSDS v2.0 (http://gsds.cbi.pku.edu.cn/) [41]. Phylogenetic tree of species was created using Taxonomy Common Tree tool at NCBI and edited and visualized using FigTree (v. 1.4.2) [38].

Ka/Ks estimation of homologous genes
Ka/Ks ratio of paralogous and orthologous FLZ genes were calculated using PAL2NAL (http:// www.bork.embl.de/pal2nal/) which is based on codeml program in PAML [42]. For Ka/Ks estimation of paralogous genes, the gene pairs from Physcomitrella patens and Selaginella moellendorffii were used. Similarly, paralogous genes from A. thaliana and Oryza sativa were identified from PLAZA v2.5 [39]. The very closely positioned proteins in the species phylogram of Picea abies and A. trichopoda were annotated as putative paralogs and used for Ka/Ks estimation. For Ka/Ks estimation of orthologous gene pairs, orthologous genes of 6 A. thaliana and O. sativa genes were identified from species occupying key taxonomic positions from Phytozome (http://www.phytozome.net/) [43]. Orthologous genes of A. thaliana genes were identified from P. patens, S. moellendorffii, A. trichopoda, Solanum lycopersicum, V. vinifera, Brassica rapa, Arabidopsis lyrata, Malus domestica, Glycine max, Populus trichocarpa and O. sativa. Orthologous genes of O. sativa genes were identified from P. patens, S. moellendorffii, A. trichopoda, Brachypodium distachyon, Hordeum vulgare, Panicum virgatum, Setaria italica, Sorghum bicolor, Z. mays and A. thaliana. The Ka/Ks ratio was estimated from each gene pair and the averaged Ka/Ks ratios were plotted in the graphs. The gene name and the Ka/Ks ratio values of each pair are given in S2 and S3 Tables.

Plant material, growth conditions and gene expression analysis
The A. thaliana Columbia (Col-0) ecotype was used for gene expression study. Seeds were surface sterilized and kept at 4°C for 48h in dark for stratification. The imbibed seeds were placed on square petri plates vertically for germination and growth. The plants were grown in 0.5X MS medium with 1% sucrose and 0.8% agar in climate-regulated growth chambers under 16:8 hours photoperiod with 22°C ± 2°C temperature and 60μmol m −2 s −1 light intensity. Samples for all seedlings stages were collected from plates and other stages were collected from plants grown in Agro peat-vermiculite (3:1) mixture at the specified climatic conditions mentioned above. The stages were determined by visible phenotype and growth stage-based phenotypic analysis of Boyes et al [47]. At least 20 plants were used for each sample and tissues were pooled together.
RNA isolation and cDNA preparation were done as described previously [48]. 1:50 diluted cDNA samples were used for qRT-PCR with SYBR-Green PCR master mix in 384-well optical reaction plates using Applied Biosystems 7500 Fast Real-Time PCR System as per manufacturer's protocol (Applied Biosystems, USA). The primers of FLZ genes were prepared from the unique region identified in each transcript using PRIMER EXPRESS version v3.0 (Applied Biosystems, USA) with default parameters. UBQ10 was used as endogenous control and mRNA level of candidate genes were calculated by 2 ΔCt method [49]. Primers used for qRT-PCR are listed in S4 Table. Results and Discussion

FLZ gene family in different genomes
In a previous study, we employed multiple strategies for the genome-wide identification of FLZ genes. A total of 757 FLZ genes were reported from 41 sequenced plant genomes [21]. Further, 8 and 43 non-redundant FLZ proteins were identified from A. trichopoda and M. acuminata genomes respectively. Additional members of FLZ gene family of R. communis, C. papaya, V. vinifera, M. truncatula and Z. mays were identified (S5 Table) [30]. It was found that the FLZ genes identified in this study also shows the general protein domain organization with single FLZ domain as observed in the previous study. Similarly, FLZ domain in the newly identified proteins also shows the typical alpha-beta-alpha topology which distinguish FLZ domain from the zf-FCS domains from bacteria, virus and metazoa [21].

Sequence conservation of FLZ proteins
It was earlier reported that the FLZ domain is highly conserved in sequence as well as secondary structure topology. To confirm the sequence conservation of FLZ domain across the plant kingdom, FLZ proteins from different species which represent key taxonomical divisions were aligned (Fig 1). It was found that apart from the conserved zinc finger signature motif CX 2 CX 17-19 FCSX 2 C, many other residues are highly conserved in the FLZ domain region across the plant kingdom which might be important in protein-protein interaction. Alignment of whole proteins identified that FLZ domain is the most conserved region in these family proteins. However, some localized conserved regions were also found in the alignment (S1 Fig). This prompted us to investigate conserved regions in the FLZ proteins other than FLZ domain. MEME search identified 15 motifs other than FLZ domain with fair degrees of conservation (S6 Table). These novel motifs were subjected to HHpred search to identify their similarity with already known domains (S7 Table). However, it was found that these motifs do not show any significant similarity with already known domains. Although these motifs have no significant relation with known domains, these domains might be evolved later and they might be contributing to the functional divergence of FLZ domain containing proteins.

Origin and expansion of FLZ genes
An extensive search in the sequenced algal genomes could not identify any FLZ genes hinting a bryophytic origin of FLZ gene family. Two FLZ genes were identified from bryophyte P. patens suggesting a bryophytic origin of FLZ genes [21]. The transition from aquatic to the terrestrial life happened due to massive changes in the genome. Comparison of P. patens genome with genomes of aquatic algae identified an increase in gene family complexity, acquisition of genes required for terrestrial life and loss of genes which are required for the survival in aquatic environments. For example, late embryogenesis abundant proteins, ABI5, HSP70 and genes involved in the processes like cytokinin, auxin, and ethylene signaling are present in P. patens which enable the plant to survive in the terrestrial environment [6]. Although the molecular functions of the FLZ gene family members in general are not well understood, many of the members are attributed to plant growth and development, stress mitigation, sugar signaling and senescence [22][23][24][25][26][27]. Based on these observations, it can be concluded that similar to the case of genes involved in stress and hormone signaling, the massive transition of habitat possibly facilitated the origin of FLZ gene also. The number of FLZ genes was found to be same in pteridophyte S. moellendorffii [21]. There is no evidence for any lineage specific WGD event in S. moellendorffii genome as the transition from a gametophyte-to a sporophyte-dominated life cycle required comparatively less acquisition of genes [50].
The number of FLZ genes was expanded to 8 in A. trichopoda and 23 in Picea abies. An ancestral seed plant specific WGD event predated the spermatophyta diversification must have contributed to the expansion of FLZ gene family in A. trichopoda and P. abies [7]. Besides this duplication, the higher expansion of FLZ gene family in P. abies compared to A. trichopoda suggests the possibility of a gymnosperm specific WGD event. However, analysis of P. abies genome does not provide evidence of any lineage specific WGD event in gymnosperms [51]. Similarly, the EST data analysis of Pinus taeda and Pinus pinaster implies the absence of Pinophyta-specific WGD events while Welwitschia mirabilis, which belongs to Gnetophyta shows an equivocal possibility of a WGD event [52]. As there is no evidence of specific WGD event in Pinophyta lineage, it can be assumed that the expansion of FLZ gene family in P. abies is possibly the result of genomic duplication events. Apart from the ancestral seed plant specific WGD event z, another WGD event predated angiosperm diversification known as ε, must also have contributed to the expansion of FLZ gene members from 2 in bryophyta and pteridophyta to 8 in A. trichopoda [3,7].The absence of lineage-specific WGD event in A. trichopoda is attributed to its reduction of specific duplication of gene families [3].This must be the reason of reduced number of FLZ genes in A. trichopoda compared to P. abies.
Phylogenetic analysis of A. trichopoda and P. abies FLZ proteins was done to delineate the evolutionary relation among the members. In the phylogram, most of the A. trichopoda FLZ proteins were found to be positioned apart suggesting high sequence divergence among them (S2A Fig). In comparison, many of the members of PabFLZ family formed clusters with short branch lengths further supports the possibility of frequent gene duplication events (S2B Fig). Besides, the slow evolutionary rate in conifers must be the reason for the formation of clusters with short branch lengths in the phylogram [51].
The number of FLZ genes is highly expanded in angiosperms [21]. In order to elucidate the evolutionary history of FLZ gene family prior to its expansion in angiosperms, comparative phylogenetic analysis was performed among P. patens, S. moellendorffii, P. abies, and A. trichopoda which are belonging to Bryophyta, Pteridophyta, Gymnospermae and Angiospermae respectively (Fig 2). The phylogram separated the proteins into distinct groups. The PpaFLZ and SmoFLZ members were found be placed distant in the phylogram implying the speciesspecific variations accumulated in FLZ genes during plant evolution. Two FLZ proteins of P. patens were positioned close in a distinct branch of phylogram. PpaFLZ1 and PpaFLZ2 showed similar exon-intron structure, protein length and domain organization implying their high similarity in sequence as well as in gene and protein structure (S4A and S4B Fig). Interestingly, the two members of S. moellendorffii displayed high divergence in both sequence and structure. Both SmoFLZ1 and SmoFLZ2 were found in the neighboring branches; however, their branch distance was found to be more than in the case of P. patens FLZ proteins (Fig 2). Most of the P. abies proteins were formed clusters with short branch lengths in the phylogram (Fig 2). These genes mainly contributed to the expansion of FLZ gene family in P. abies. Considering the absence of WGD in Pinophyta, this observation implies the pivotal role of genomic block duplications in the expansion of FLZ gene family in P. abies. Among the A. trichopoda FLZ proteins, AtrFLZ3 was found to be in a distinct branch more close to P. patens FLZ proteins suggesting that this protein is very similar to the ancestral FLZ proteins. AtrFLZ5, AtrFLZ6, AtrFLZ7, and AtrFLZ8 formed a distinct clade; however, the branch length was found to be higher among these proteins (Fig 2). The remaining three A. trichopoda proteins were found to be positioned close to P. abies proteins suggesting that these proteins are more related to ancestral FLZ proteins.
The exon-intron structure was found to be considerably conserved among most of the PabFLZ genes while comparatively more variation in the intron length was observed among AtrFLZ genes (S4A Fig). All these results suggest that individual genes accumulated more sequence as well as structural variation in A. trichopoda. In the case of P. abies, genomic block duplication played a major role in the expansion of this gene family and the duplicated genes were seen to be very close in the phylogram. The slow evolutionary rate in the conifers must be contributed to this scenario. At the functional level, this suggests the possibility of increased amount of functional redundancy among closely related members in P. abies.
In order to analyse the evolutionary expansion of FLZ genes in angiosperms, the duplication data of FLZ genes from selected species was retrieved from PLAZA v2.5 [35]. It was noticed that the increased number of FLZ genes in many species is the result of lineage-specific block and tandem duplications ( Table 1). The similar number and duplication history of FLZ genes in O. sativa, B. distachyon and S. bicolor suggest that the expansion of FLZ genes in these species is the result of whole genome duplication predated diversification of Poaceae or before that. The ancient monocot specific τ duplication and σ and ρ duplications which were occurred after the divergence of Poales from Liliales, Zingiberales and Arecales must have contributed to the similar number and duplication history of FLZ gene family in these Poaceae species [9,10]. A lineage-specific WGD event in Z. mays which distinguish this species from its closest relative S. bicolor, lead to increasing the number of FLZ genes to 47 (S5 Table) [53]. Apart from the monocot specific τ duplication, the γ duplication event common to Zingiberales and α and β duplications specific to Musa lineage must be the reason for the high expansion of FLZ genes in M. acuminata [9,10].
The evolutionary pattern of FLZ genes in the sequenced dicot species shows lineage-specific variations. The Carica papaya genome contains 10 non-duplicated FLZ genes. A genome triplication prior to core eudicot radiation and the subsequent gene loss must have restricted the increase of FLZ gene number from 8 in A. trichopoda to 10 in this species [1]. The V. vinifera and C. papaya genomes have not undergone any recent lineage-specific WGD event [15,54]. However, the number of FLZ genes increased to 14 in V. vinifera which can be the result of genomic block duplication events (S5 Table). The expansion of FLZ genes to 12 in Theobroma cacao can be the product of one round of genomic block duplication in two ancestral genes (Table 1). FLZ genes in A. thaliana, which is another member in eurosid II, underwent more duplication. Two lineage-specific WGD after their division from eurosid I and subsequent gene loss and extensive genomic block duplications shaped A. thaliana genome [4,[55][56][57]. This could be the reason for the comparatively higher occurrence of duplicated genes in FLZ gene family of A. thaliana with no major leap in the numbers despite two lineage-specific WGD events. Analysis of the evolutionary history of FLZ genes in the malpighiales order of eurosid I clade also confirms the role of duplication and gene loss in the expansion of this gene family. The increase in FLZ gene number and higher representation of duplicated genes were observed in P. trichocarpa and Manihot esculenta while only two events of block duplication were observed in R. communis. The expansion of FLZ gene number in P. trichocarpa can be attributed to the salicoid specific WGD that happened after the divergence of eurosid clades [11]. The absence of lineage-specific WGD can be accounted for the lack of greater expansion of FLZ gene family in R. communis [16]. M. esculenta, which is another member of the family euphorbiaceae showed a comparatively larger expansion of FLZ gene family than in the case of R. communis. Although the genome of M. esculenta is not published yet, the increased number and high representation of duplicated genes in the FLZ gene family could predict a lineage specific WGD event in Manihot. Fragaria vesca genome contains 14 FLZ genes. Three rounds of genomic block duplication events in F. vesca must have contributed in the expansion of FLZ genes to 14 in this species (Table 1). The absence of WGD in the Fragaria lineage can be accounted for the less number of FLZ genes in F. vesca compared to other rosaceae species, M. domestica [58]. A WGD event 30-45 MYA ago after the divergence from Fragaria must be contributed to the expansion of FLZ gene family in M. domestica [12]. In legumes, the high expansion of FLZ genes in G. max must be due to a lineage-specific WGD event that occurred about 13 million years ago [13]. The absence of large-scale duplication events in the galegoid tribe restricted their number in Lotus japonicus [2]. However, the number for FLZ gene family is found to be high in M. truncatula. The high rate of genomic block duplications increased gene content and expansion of many gene families in this species [17]. This can be accounted for the expansion of FLZ gene family in M. truncatula despite the absence of lineage-specific WGD events.
Analysis of evolutionary expansion of FLZ gene family in spermatophyta clearly implies the role of WGD events. The general and lineage-specific WGD events reported were found to be correlated with the expansion of FLZ gene family (S3 Fig). Besides, the FLZ gene family size and duplication history of M. esculenta indicate the possibility of a WGD event in the Manihot lineage. Interestingly, analysis of myosin gene family also predicts a WGD event in Manihot lineage [59]. From the 8 members in A. trichopoda, the genome triplication common to eudicots could only marginally increase the number of FLZ genes in species with no lineage-specific WGD such as C. papaya, V. vinifera, L. japonicus etc suggesting a high rate of gene loss after this genome triplication. A recent gene family size comparison of Nucleobase-Ascorbate Transporter (NAT) gene family among eudicots yielded similar observation. The number of NAT genes increased from 7 in A. trichopoda to only 9 in V. vinifera and C. papaya despite the basal eudicot genome triplication [60]. Among monocots, the WGD predated to the diversification of species studied lead to the expansion of FLZ gene family in general while lineage-specific WGD in M. acuminata and Z. mays lead to increased number of genes in these two species. In genomes like M. truncatula and P. abies, genomic block duplication was found to be the major contributing factor in the expansion of FLZ gene family.

Phylogenetic analysis of Arabidopsis thaliana FLZ gene family
A. thaliana genome possesses 18 FLZ genes which are distributed in all 5 chromosomes (Fig 3). FLZ genes were found in all chromosomes; however, segment-specific duplications resulted in unequal distribution of genes. Among 18 members, only 3 members i.e., AthFLZ7, AthFLZ15, and AthFLZ16 did not undergo any duplication. AthFLZ17 and AthFLZ18 are products of tandem duplication with no divergence in sequence. All other members are products of block duplication events. As discussed earlier, the A. thaliana is an extensively duplicated genome with subsequent gene loss and differential radiation of many gene families [4].
In order to delineate the evolutionary history of individual A. thaliana FLZ genes, a phylogram of FLZ proteins was constructed along with members of P. patens, S. moellendorffii and A. trichopoda (Fig 4). As observed in the phylogram of P. patens, S. moellendorffii, P. abies, and A. trichopoda, the P. patens proteins were found in a distinct branch. Similarly, AtrFLZ3 was found in a distinct branch more close to P. patens proteins. The S. moellendorffii FLZ proteins were found in separate branches confirming the sequence divergence accumulated in Pteridophyta. The non-duplicated AthFLZ7 was close to SmoFLZ2 while SmoFLZ1 was found close to another non-duplicate AthFLZ15 and A. trichopoda members AtrFLZ6, AtrFLZ7, and AtrFLZ8. As observed in the earlier phylogram, AtrFLZ1 and AtrFLZ2 were found close in a distinct branch. The remaining two A. trichopoda proteins were found close to the duplicated A. thaliana FLZ proteins suggesting that these members are related to the ancestral FLZ genes which had undergone duplication in A. thaliana.
The gene structure, transcript forms and protein domain organization of A. thaliana FLZ gene family were studied to analyse the structural changes happened in the genes during the course of evolution (S5 Fig). The gene organization was found to be conserved in most cases. However, change in the length of exon, intron and UTRs was observed frequently. Except AthFLZ12 and AthFLZ16, all other genes possess 5' and 3' UTRs and two exons. AthFLZ12 Comparison of the gene structure of duplicated genes revealed structural divergence in few paralogous pairs (S5A Fig). The tandem duplicated AthFLZ17 and AthFLZ18 genes showed no difference in the structural organization. In case of block duplicated paralogs AthFLZ1, AthFLZ2 and AthFLZ3, variation was observed in the length of UTRs, intron and exons while no variation in the structural organization was observed. The same feature was observed in the case of block duplicated AthFLZ5-AthFLZ6 and AthFLZ10-AthFLZ11 gene pairs. Along with variation in the length of gene components, divergence in the gene organization was observed in AthFLZ8-AthFLZ9 and AthFLZ12-AthFLZ13 block duplicated gene pairs. The changes in the exon length resulted in the divergence of protein length in many duplicated genes (S5C Fig). Phylogenetic analysis of Oryza sativa FLZ gene family O. sativa genome contains 29 FLZ genes which were found to be distributed in all chromosomes except chromosome number 12 (Fig 5). The O. sativa homologue of AthFLZ1 was named as OsaFLZ1 and the remaining genes were named according to its closeness with this gene in the protein phylogram (S6 Fig). Due to segment-specific block duplication events, the chromosomal distribution of FLZ gene family in O. sativa was found to be more unequal than in the case of A. thaliana. Analysis of O. sativa genome identified a high representation of tandem repeats of genes [61]. Consistent with this general feature of the genome, more representation of tandem duplicated genes was found in O. sativa FLZ gene family compared to A. thaliana. However, the percentage of non-duplicated genes was found to be higher in O. sativa compared to A. thaliana ( Table 1). The high incidence of block and tandem duplication happened in chromosomes 2 and 4 is one of the reasons behind the increased number of FLZ genes in O. sativa. As discussed earlier, the monocot specific σ and Poales specific ρWGD events also contributed to the increase of FLZ genes in grasses including rice [8,9].
A phylogram was constructed with O. sativa, P. patens, S. moellendorffii, and A. trichopoda FLZ gene family members to analyse the evolutionary history of O. sativa genes (Fig 6). The extensively duplicated genes in the chromosome 2 and 4 formed a distinct cluster with large branches. AtrFLZ4 was also found in this cluster suggesting that this gene is close to the ancestral gene which underwent duplication. Similarly, most of the A. trichopoda proteins were found in distinct clusters with O. sativa proteins suggesting their similarity with the ancestral genes in monocots which underwent duplication during the course of time.
Phylogenetic comparison of O. sativa FLZ gene family with P. patens, S. moellendorffii, and A. trichopoda FLZ gene families revealed the evolutionary history of FLZ genes in O. sativa. The phylogenetic analysis further confirms two hypotheses proposed earlier. Firstly, the Bryophyta-Pteridophyta divergence in the FLZ genes is also evident in the analysis of O. sativa FLZ  OsaFLZ25.2 and OsaFLZ27.2 lack FLZ domain completely. OsaFLZ22.1 have functional FLZ domain while other two splice variants possess truncated FLZ domain with first two cysteine residues. It is already found that the FLZ domain with all four conserved cysteine residues intact is important for protein-protein interaction [22]. FLZ domain is the only conserved functional domain in O. sativa FLZ gene family. Taking this fact into account, the biological functions of splice variants without FLZ domain or truncated domain has to scrutinize further.
The gene structures of duplicated genes were compared to identify the structural divergence. Block duplicated pairs OsaFLZ13-OsaFLZ14 and OsaFLZ28-OsaFLZ29 have similar structural organization albeit little variation in the length of gene components (S7A Fig). High divergence in the gene structure was observed in the duplicated genes in the segment of chromosomes 2 and 4. OsaFLZ1 displays the typical two exon pattern with both UTRs and a large intron.
OsaFLZ3 also have the same gene structure with a shorter intron while another paralog, OsaFLZ2 have shorter intron but lack both UTRs (S7A Fig). Similarly, structural variation was also observed in duplicated genes OsaFLZ4, OsaFLZ5, and OsaFLZ6. OsaFLZ4 possess a larger intron but lack both UTRs while OsaFLZ5 was found to be intronless and carry both UTRs. OsaFLZ6 shows typical FLZ gene pattern with a shorter intron. In block duplicated pair OsaFLZ7 and OsaFLZ8, the latter has an intron in 3'UTR. OsaFLZ18 found to have two introns in 5'UTR while its paralogue OsaFLZ17 has only one intron in 5'UTR. Similarly, gain and loss of gene components was also observed in block duplicates OsaFLZ19 and OsaFLZ20.

Analysis of evolutionary pressure on FLZ gene family
The duplicated genes undergo selection processes like hypofunctionalization, neofunctionalization, subfunctionalization and nonfunctionalization according to the evolutionary pressure. The survivors of the selection pressure undergo strong purifying selection [62]. The Ka/Ks ratio is a widely used method to analyse the selection pressure acting on protein-coding genes [63]. The Ka/Ks ratio of the FLZ gene pairs of P. patens and S. moellendorffii, putative duplicates of P. abies and A. trichopoda and duplicated gene pairs of A. thaliana and O. sativa were estimated (Fig 7A). Except in P. abies, the Ka/Ks ratios of FLZ genes were found to be very much less than1 indicating a strong purifying selection (Fig 7A and 7B). Although the Ka/Ks ratio was found to be less than 1 n all gene pairs of P. abies except in PabFLZ14-PabFLZ15, these gene pairs showed higher ratio value when compared to other species studied. The lower nucleotide substitution rate compared to angiosperms due to their comparatively long life span is a characteristic feature of conifer genome evolution [51,64,65]. This feature of conifers explains the increased Ka/Ks ratio of FLZ genes observed in P. abies. Further, the Ka/Ks ratios of orthologous FLZ gene pairs were also studied. Six genes from A. thaliana and O. sativa each were selected and Ka/Ks ratio with their orthologous genes from selected species were estimated (S2 and S3 Tables). The Ka/Ks ratios of orthologous gene pairs were also found to be lower than 1 confirming the strong purifying selection in FLZ gene family (Fig 7C and 7D).
It is an already established fact that the selection pressure acting on a gene is not uniform on the entire stretch. Different modules can be under different selection pressure creating a chimera of selection pressure on a single gene. In case of FLZ proteins, it is already found that the FLZ domain part is the most conserved module in the whole protein (S1 Fig). Taking all these information into account, we calculated the Ka/Ks ratio of domain region of paralogous FLZ gene pairs (Fig 7A). In general, a significant reduction in the Ka/Ks ratio was observed in FLZ domain region when compared to whole protein (Fig 7A and 7B). However, in some cases such as AtrFLZ1-AtrFLZ2, AthFLZ1-AthFLZ2, and OsaFLZ1-OsaFLZ2, the ratio was found higher for the domain region compared to whole protein. These results suggest that generally the FLZ domain region is under more purifying selection and in some cases whole protein was found to be under more strict purifying selection. In general, these results confirm that the evolutionary pressure on FLZ gene is not uniform on the entire stretch.

Spatiotemporal expression analysis of Arabidopsis thaliana FLZ gene family
Understanding the spatiotemporal expression dynamics of genes can give clues about their function. Microarray-based expression analysis of A. thaliana FLZ gene family revealed that FLZ gene family have a varied expression pattern. Many of the FLZ gene family members found to have induced expression in reproductive organs and silique [22]. However, a detailed developmental-and organ-specific expression study of FLZ gene family is lacking. In this study, we analysed the expression pattern of 18 A. thaliana FLZ genes in different developmental stages and organs using qRT-PCR (Fig 8).
Most of the FLZ genes showed increased expression in flower and/or silique stages suggesting a specific function in the development of these organs. AthFLZ1 expressed high in developing seed, flower etc compared to seedling stages and vegetative organs. Among the paralogous genes of AthFLZ1, AthFLZ2 showed similar expression pattern while AthFLZ3 showed induced expression in flower, silique and seedling stages suggesting divergence in its expression domain. The block duplicated pair AthFLZ5 and AthFLZ6 showed overlapping expression  domain with a small amount of divergence. AthFLZ7 was found to be profusely expressed in seedling stages, root and flower and mature silique. Similarly, AthFLZ9 also showed high induction of expression in open flower compared to flower bud. AthFLZ8 was found to be highly expressed in developing seedlings. AthFLZ10 showed a uniform expression pattern in all stages studied except root and leaf of mature rosette where it showed highly reduced expression. The homologous AthFLZ11 gene showed almost similar expression pattern with a fair degree of divergence. Another block duplicated pair, AthFLZ12 and AthFLZ13 showed high divergence in expression domain. AthFLZ12 was expressed uniformly in all tissues studied except open flower and mature silique where it showed high expression compared to other tissues. However, the homologue AthFLZ13 showed higher expression in both flower and silique stages studied. Besides, AthFLZ13 also showed reduced expression in root and leaf of bolted plant. Unlike the majority of other FLZ genes, AthFLZ14 showed higher expression in seedling stages compared to reproductive organs. AthFLZ15 showed specific expression in reproductive organs. The expression of AthFLZ16 was highly up-regulated in open flower and mature silique. The tandem duplicated pair AthFLZ17/18 showed almost uniform expression pattern in vegetative stages while expression was found to be highly up-regulated in reproductive stages.
Gene expression analysis from publically available microarray data revealed that A. thaliana FLZ gene family is highly responsive to hormonal and nutritional cues. Most of the A. thaliana FLZ genes are up-regulated in response to glucose treatment [22]. Consistent with this, the interaction of FLZ proteins with RAPTOR1B, SnRK1.1 and SnRK1.2 confirms the involvement of FLZ gene family in sugar and energy signaling [22,27]. The role of sugars in promoting vegetative-to-reproductive phase transition and promoting flowering is well documented [66][67][68][69]. Sugars, through HEXOKINASE1-dependent signaling, repress microRNA156 at transcriptional and post-transcriptional levels which release the inhibition of key regulators of juvenileto-adult and vegetative-to-reproductive phase transition like SQUAMOSA PROMOTER BINDING PROTEIN-LIKE transcriptional factors [66,67].
As the role of FLZ genes in sugar and energy signaling is emerging, we tried to explore the possibility of involvement of FLZ gene in vegetative-to-reproductive phase transition through analysis of expression dynamics of these genes during the period. Four rosette growth stages based on growth stage-based phenotypic analysis of Boyes et al. and according to visible phenotype were selected for the analysis of expression pattern of FLZ genes [47]. At 22 days and 26 days, the rosette attained approximately 50% and 70% of final rosette size respectively without any visible flower bud appearance. The rosette attained full size at 28 days with appearance of flower buds, but no bolting. At 32 days, the plants displayed 1 cm length bolt with prominent flower bud appearance. The expression of all 18 FLZ genes in these four developmental stages was analysed by qRT-PCR (Fig 9). Interestingly, all genes showed fluctuations in expression in the stages studied. In many genes, compared to 22 days old rosette, the expression was up-regulated in 26 days old rosette which was the prerequisite stage of flower bud appearance. Different expression dynamics were observed after the induction of expression from 22 days to 26 days. The increased expression in 26 days old was found to be gradually declined in other stages in most of the genes. In the case of AthFLZ1, the increased expression was more or less retained in other stages. AthFLZ9 showed decline of expression in 28 days while the expression was got induced in 32 days. This preliminary analysis gives clues about the role of FLZ gene in vegetative-to-reproductive phase transition. Considering the role of sugar in phase transition in graph is as follows, RS, radicle emerged; CS, cotyledon stage seedling; LS, 2 leaves stage seedling; MR, mature plant root; ML, mature bolts rosette leaf; FB, flower bud; FO, flower open; SS, silique small; SM, silique mature. doi:10.1371/journal.pone.0134328.g008 and the recent integration of FLZ gene family in sugar and energy signaling, this observation invites an extensive molecular dissection.
Analysis of expression pattern of A. thaliana FLZ gene family revealed that the expression of the majority of FLZ genes is induced in reproductive organs compared to vegetative stages and organs. This organ-specific induction of FLZ gene family suggests a possible role of this gene family in controlling reproductive development. It was also found that most of the FLZ genes display a similar expression dynamics during the vegetative-to-reproductive phase Fig 9. Expression analysis of Arabidopsis thaliana FLZ gene family during phase transition and bolting using real-time PCR. UBQ10 is used as endogenous control. The absolute expression is calculated from ΔCT value. Graphs represent average and error bars represent mean ± SD of two biological replicates. X axis represent samples and Y axis represent absolute expression value. Samples are abbreviated in graph is as follows, 22d, rosette 22 day old; 26d, rosette 26 day old; 28d, rosette 28 day old; 32d, bolted rosette 32d day old. Comprehensive Analysis of FLZ Gene Family transition. The common expression domains displayed by even diverged FLZ genes suggest the possibility of a high degree of functional conservation in this gene family. As it is observed in the case of many multigene families, the strong purifying selection observed in the FLZ gene family must be responsible for the functional redundancy. Consistent with this hypothesis, functional redundancy aspect of FLZ gene family is also revealed during protein-protein interaction studies. It is already found that FLZ protein share many common interacting partners such as RAPTOR1B, SnRK1.1, SnRK1.2 which are conserved regulators of plant growth [27]. In a detailed protein-protein interaction study with FLZ proteins, it was identified that all 18 A. thaliana FLZ can interact with SnRK1.1 and SnRK1.2 which are the kinase subunits of SnRK1 [22]. Both SnRK1 and TOR are multi-subunit protein kinases and the interaction of many FLZ proteins with subunits of these kinases suggest the possibility that may be FLZ proteins also form complex with these kinases in response to different cues. The common expression domain shared by many of the FLZ genes might be facilitating this complex formation during different stages of plant development.

Analysis of expression pattern divergence of duplicated Arabidopsis thaliana FLZ genes
Large-scale expression analysis of A. thaliana and O. sativa duplicated genes identified that in many cases paralogs underwent divergence in the expression domain [70][71][72]. Analysis of expression pattern divergence in duplicated genes serves as an excellent tool to study the functional divergence in the gene family. From the qRT-PCR based expression data of FLZ genes, the expression profile of 5 block duplicated gene pairs were analysed for expression pattern divergence (S8 Fig). Comparison of expression pattern of AthFLZ1 and AthFLZ2 identified a high degree of expression retention in this block duplicated pair. AthFLZ1 and AthFLZ2 displayed comparatively shorter branch length in the protein phylogram of A. thaliana (Fig 4). Comparison of expression profile of AthFLZ5 and AthFLZ6 identified divergence in expression domain. The expression of AthFLZ5 was found to be restricted to the reproductive organs while there was a significant amount of AthFLZ6 expression in seedling stages also. No divergence in expression was observed during vegetative-to-reproductive phase transition stages. A high degree of expression divergence was observed in the expression pattern of AthFLZ8 and AthFLZ9 in seedling stages, reproductive organs and vegetative-to-reproductive phase transition stages. AthFLZ8 and AthFLZ9 also displayed a very high degree of sequence divergence in the protein phylogram (Fig 4). A fair degree of expression divergence was also observed in the AthFLZ10-AthFLZ11 and AthFLZ12-AthFLZ13 block duplicated gene pairs.
The analysis of expression divergence among block duplicated A. thaliana FLZ genes revealed branch specific variation in the degree of expression divergence. AthFLZ1-AthFLZ2 gene pair showed a high degree of expression retention. Similarly, many of other gene pairs also showed expression retention in many developmental stages and tissues studied. This indicates the possible functional redundancy among duplicated genes. The strong purifying selection observed in this gene family must be a contributing to the functional redundancy of duplicated genes. As observed in the case of AthFLZ8-AthFLZ9 gene pair, few genes displayed unique and sometimes contrasting expression pattern compared to its paralogous gene. This can be because of gain or loss of expression domains after duplication. A positive correlation between expression divergence and sequence and/or structural divergence among duplicated genes is observed in many species such as yeast, human, and Caenorhabditis elegans [73]. However, in the case of A. thaliana, different groups identified positive, negative and zero correlations [70,71,73,74] which suggest that the correlation between sequence and expression divergence can be varied in different duplicated genes. In FLZ gene family, the duplicated gene pairs with high sequence divergence showed a high degree of expression divergence while the pairs with less sequence divergence showed comparatively less degree of expression divergence. This observation suggests that the correlation between sequence and expression divergence in the duplicated genes in A. thaliana FLZ gene family is most likely positive.

Conclusions
The present study gives a comprehensive account on the evolution of FLZ gene family and the expression dynamics of A. thaliana FLZ gene family in different developmental stages and tissues. The FLZ domain was found to be highly conserved across the plant kingdom. Apart from FLZ domain, MEME search identified novel motifs in FLZ proteins which must be contributing to the functional divergence of this family. Comparison of WGD events happened in plant lineage and FLZ gene family size revealed that general-and lineage specific WGD events and subsequent gene loss etc contributed in the evolutionary expansion of this family. Genomic duplication events also contributed to the expansion of FLZ gene family in many cases. Detailed analysis of FLZ gene family of A. thaliana and O. sativa revealed monocot and dicot specific variations in the evolution of this gene family. The FLZ genes were found to under high purifying selection. The expression analysis of A. thaliana FLZ gene family suggests their role in vegetative development, vegetative-to-reproductive phase transition and reproductive development. The common interacting proteins identified before and common expression domains revealed in the study imply functional redundancy in FLZ gene family. This study will be helpful in uncovering the functions played by the FLZ gene family in plant growth and development which is not yet completely understood.