Evolution of the PEBP gene family in plants: functional diversification in seed plant evolution

The phosphatidyl ethanolamine-binding protein (PEBP) gene family is present in all eukaryote kingdoms, with three subfamilies identified in angiosperms (FLOWERING LOCUS T [FT], MOTHER OF FT AND TFL1 [MFT], and TERMINAL FLOWER1 [TFL1] like). In angiosperms, PEBP genes have been shown to function both as promoters and suppressors of flowering and to control plant architecture. In this study, we focus on previously uncharacterized PEBP genes from gymnosperms. Extensive database searches suggest that gymnosperms possess only two types of PEBP genes, MFT-like and a group that occupies an intermediate phylogenetic position between the FT-like and TFL1-like (FT/TFL1-like). Overexpression of Picea abies PEBP genes in Arabidopsis (Arabidopsis thaliana) suggests that the FT/TFL1-like genes (PaFTL1 and PaFTL2) code for proteins with a TFL1-like function. However, PaFTL1 and PaFTL2 also show highly divergent expression patterns. While the expression of PaFTL2 is correlated with annual growth rhythm and mainly confined to needles and vegetative and reproductive buds, the expression of PaFTL1 is largely restricted to microsporophylls of male cones. The P. abies MFT-like genes (PaMFT1 and PaMFT2) show a predominant expression during embryo development, a pattern that is also found for many MFT-like genes from angiosperms. P. abies PEBP gene expression is primarily detected in tissues undergoing physiological changes related to growth arrest and dormancy. A first duplication event resulting in two families of plant PEBP genes (MFT-like and FT/TFL1-like) seems to coincide with the evolution of seed plants, in which independent control of bud and seed dormancy was required, and the second duplication resulting in the FT-like and TFL1-like clades probably coincided with the evolution of angiosperms.     

A promoter analysis of MOTHER OF FT AND TFL1 1 (JcMFT1), a seed-preferential gene from the biofuel plant Jatropha curcas

MOTHER OF FT AND TFL1 (MFT)-like genes belong to the phosphatidylethanoamine-binding protein (PEBP) gene family in plants. In contrast to their homologs FLOWERING LOCUS T (FT)-like and TERMINAL FLOWER 1 (TFL1)-like genes, which are involved in the regulation of the flowering time pathway, MFT-like genes function mainly during seed development and germination. In this study, a full-length cDNA of the MFT-like gene JcMFT1 from the biodiesel plant Jatropha curcas (L.) was isolated and found to be highly expressed in seeds. The promoter of JcMFT1 was cloned and characterized in transgenic Arabidopsis. A histochemical β-glucuronidase (GUS) assay indicated that the JcMFT1 promoter was predominantly expressed in both embryos and endosperms of transgenic Arabidopsis seeds. Fluorometric GUS analysis revealed that the JcMFT1 promoter was highly active at the mid to late stages of seed development. After seed germination, the JcMFT1 promoter activity decreased gradually. In addition, both the JcMFT1 expression in germinating Jatropha embryos and its promoter activity in germinating Arabidopsis embryos were induced by abscisic acid (ABA), possibly due to two ABA-responsive elements, a G-box and an RY repeat, in the JcMFT1 promoter region. These results show that the JcMFT1 promoter is seed-preferential and can be used to control transgene expression in the seeds of Jatropha and other transgenic plants.     

Cloning and Characterization of FLOWERING LOCUS T-Like Genes from the Perennial Geophyte Saffron Crocus (Crocus sativus)

The transition to flowering is one of the most important developmental decisions made by plants. At the molecular level, many genes coordinate this transition. Among these, genes encoding for phosphatidylethanolamine-binding proteins (PEBPs) play important roles in regulating flower time and the fate of inflorescence meristem. To investigate the role of PEBPs in an industrially important crop cultivated for its nutritional and medicinal properties, the monocotyledonous species Crocus sativus L., we have isolated three FLOWERING LOCUS T (FT)-like genes designated as CsatFT1-like, CsatFT2-like, and CsatFT3-like. The isolated genes maintain the exon/intron organization of FT-like genes and encode proteins similar to the members of the PEBP family. Phylogenetic and amino acid analysis at critical positions confirmed that the isolated sequence belongs to the FT clade of the PEBP family phylogeny distinctly from the TERMINAL FLOWER 1 (TFL1) and MOTHER OF FT AND TFL1 clades. Expression analysis indicated differences in the expression of the three FT-like genes in different organs and different expressions during the day–night diurnal clock. Additionally, analysis of isolated promoter sequences using computational methods reveals the preservation of common binding motifs in FT-like promoters from other species, thus suggesting their importance among plant species.     

Characterization and expression analysis of TERMINAL FLOWER1 homologs from cultivated alloteraploid cotton (Gossypium hirsutum) and its diploid progenitors

The seasonal cycle and persistence of a plant is governed by a combination of the determinate or indeterminate status of shoot and root apical meristems. A perennial plant is one in which the apical meristem of at least one of its shoot axes remains indeterminate beyond the first growth season.TERMINAL FLOWER1 (TFL1) genes play important roles in regulating flowering time, the fate of inflorescence meristem and perenniality. To investigate the role of TFL1-like genes in the determination of the apical meristems in an industrially important crop cultivated for its fibers, we isolated and characterized two TFL1 homologs (TFL1a and TFL1b) from tetraploid cultivated cotton (Gossypium hirsutum) and its diploid progenitors (Gossypium arboreum and Gossypium raimondii). All isolated genes maintain the same exon–intron organization. Their phylogenetic analysis at the amino acid level confirmed that the isolated sequences are TFL1-like genes and collocate in the TFL1 clade of the PEBP protein family. Expression analysis revealed that the genes TFL1a and TFL1b have slightly different expression patterns, suggesting different functional roles in the determination of the meristems. Additionally, promoter analysis by computational methods revealed the presence of common binding motifs in TFL1-like promoters. These are the first reported TFL1-like genes isolated from cotton, the most important crop for the textile industry.     

PEATmoss (Physcomitrella Expression Atlas Tool): a unified gene expression atlas for the model plant Physcomitrella patens

Physcomitrella patens is a bryophyte model plant that is often used to study plant evolution and development. Its resources are of great importance for comparative genomics and evo‐devo approaches. However, expression data from Physcomitrella patens were so far generated using different gene annotation versions and three different platforms: CombiMatrix and NimbleGen expression microarrays and RNA sequencing. The currently available P. patens expression data are distributed across three tools with different visualization methods to access the data. Here, we introduce an interactive expression atlas, Physcomitrella Expression Atlas Tool (PEATmoss), that unifies publicly available expression data for P. patens and provides multiple visualization methods to query the data in a single web‐based tool. Moreover, PEATmoss includes 35 expression experiments not previously available in any other expression atlas. To facilitate gene expression queries across different gene annotation versions, and to access P. patens annotations and related resources, a lookup database and web tool linked to PEATmoss was implemented. PEATmoss can be accessed at https://peatmoss.online.uni-marburg.de     

BROTHER OF FT AND TFL1 (BFT), a member of the FT/TFL1 family,shows distinct pattern of expression during the vegetative growth of Arabidopsis

Transition to the flowering stage is precisely controlled by a few classes of regulatory molecules. BROTHER OF FT AND TFL1 (BFT) is a member of FLOWERING LOCUS T (FT)/TERMINAL FLOWER 1 (TFL1) family, an important class of flower development regulators with unidentified biochemical function. BFT has a TFL1-like activity and plays a role in axillary inflorescence development. To elucidate the expression pattern of BFT, we analyzed the subcellular localization and conditional expression of BFT in this study. We generated 35S::BFT:GFP plants to investigate the subcellular localization of BFT protein. 35S::BFT:GFP plants showed late flowering, similarly as did 35S::BFT plants. BFT:GFP fusion protein was localized in the nucleus and the plasma membrane, which was different from the localization pattern of FT and TFL1. BFT expression was induced by abiotic stress conditions. ABA, drought, and osmotic stress treatments induced BFT expression, whereas cold, salt, and heat stress conditions did not, suggesting that BFT plays a role in regulating flowering time and inflorescence structure under drought conditions. The induction pattern of BFT was different from those of other FT/TFL1 family genes. Our studies indicated that BFT showed a distinct expression pattern from its homologous genes during the vegetative growth in Arabidopsis.Key words: flowering time, flowering locus T, terminal flower 1, brother of FT and TFL1, abiotic stress, subcellullar localizationThe FLOWERING LOCUS T (FT)/TERMINAL FLOWER 1 (TFL1) family is a small gene family whose members play a pivotal role in flower development in Arabidopsis. The family includes FT, TFL1, TWIN SISTER OF FT (TSF), Arabidopsis thaliana CENTRORADIALIS homologue (ATC), MOTHER OF FT AND TFL1 (MFT) and BROTHER OF FT AND TFL1 (BFT).^{3,5,6,9,15,17} FT is a floral promoter that integrates signal inputs from various pathways that regulate flowering time in Arabidopsis.^5,6 TFL1 plays an antagonistic role to that of FT, functioning as a floral inhibitor. Unlike FT, TFL1 also plays an important role in controlling plant architecture by regulating the expression of LEAFY (LFY) and APETALA1 (AP1), two important floral meristem identity genes in the shoot apical meristem (SAM).^3,7 TSF regulates flowering by a mechanism similar to that of FT, although a lesion in TSF does not have an apparent effect on the determination of flowering time. MFT has a weak FT-like activity.¹⁷ ATC acts as a floral repressor, and its role is similar to that of TFL1.⁹ Finally, BFT has a TFL1-like activity, in spite of its amino acid homology to FT,^2,4,16 and functions redundantly with TFL1 in inflorescence meristem development in Arabidopsis.¹⁶ Although genetic studies elucidated an intricate role of the FT/TFL1 family genes, not much is known about the expression pattern of the remaining members except FT and TFL1. Here, we report that BFT expression showed a distinct pattern from its homologous genes during the vegetative phase. BFT:GFP fusion protein was detected in the nucleus and the plasma membrane. BFT expression was induced by abiotic stress conditions, distinct from other FT/TFL1 family genes, raising the possibility that BFT plays a role in regulating flowering time and inflorescence structure under drought conditions.     

Bioinformatics search for plant homologues of Ste20-like serine/threonine protein kinases

Eleven plant homologuess of animal and yeast STE20-like protein kinases were identified. It was shown that unknown proteins A9RVK0 of the moss Physcomitrella patens ssp. patens and A7P2E2 of the grape Vitis vinifera were the closest plant homologues of STE20-like protein kinases. The Ste20-like protein kinase dst1 of the myxomycete Dictyostelium discoideum was the closest to the plant homologues. The spatial structure of the catalytic domain of the protein A9RVK0 from P. patens ssp. patens was predicted.     

Functional characterization of LIKE HETEROCHROMATIN PROTEIN 1 in the moss Physcomitrella patens: its conserved protein interactions in land plants

In flowering plants, LIKE HETEROCHROMATIN PROTEIN 1 (LHP1)/TERMINAL FLOWER 2 (TFL2) is known to interact with polycomb group (PcG) and non‐PcG proteins and control developmental programs. LHP1/TFL2 is an ancient protein and has been characterized in the early‐divergent plant Physcomitrella patens. However, interacting partners of PpLHP1 other than the chromomethylase PpCMT have not been identified to date. Also, while functional polycomb repressive complex 2 (PRC2) is known to exist in P. patens, there is no experimental evidence to support the existence of PRC1‐like complexes in these mosses. In this study, using protein?protein interaction methods, transient expression assays and targeted gene knockout strategy, we report the conserved properties of LHP1/TFL2 using the Physcomitrella system. We show that a PRC1‐like core complex comprising of PpLHP1 and the putative PRC1 Really Interesting New Gene (RING)‐finger proteins can form in vivo. Also, the interaction between PpRING and the PRC2 subunit PpCLF further sheds light on the possible existence of combinatorial interactions between the Polycomb Repressive Complex (PRC) in early land plants. Based on the interaction between PpLHP1 and putative hnRNP PpLIF2‐like in planta, we propose that the link between PpLHP1 regulation and RNA metabolic processes was established early in plants. The conserved subnuclear distribution pattern of PpLHP1 in moss protonema further provides insight into the manner in which LHP1/TFL2 are sequestered in the nucleoplasm in discrete foci. The PpLHP1 loss‐of‐function plants generated in this study share some of the pleiotropic defects with multiple aberrations reported in lhp1/tfl2. Taken together, this work documents an active role for PpLHP1 in epigenetic regulatory network in P. patens.     

The FLOWERING LOCUS T/TERMINAL FLOWER 1 family in Lombardy poplar

Genes in the FLOWERING LOCUS T (FT) and TERMINAL FLOWER 1 (TFL1)family have been shown to be important in the control of theswitch between vegetative and reproductive growth in severalplant species. We isolated nine members of the FT/TFL1 familyfrom Lombardy poplar (Populus nigra var. italica Koehne). Sequenceanalysis of the members of the FT/TFL1 family revealed considerablehomology within their coding regions both among family membersand to the members of the same family in Arabidopsis, tomatoand grapevine. Moreover, members of this family in all fourspecies examined display a common exon–intron organization.Phylogenetic analysis revealed that the genes fall into fourdifferent clades: two into the TFL1 clade; five into the FTclade; and one each into the MOTHER OF FT AND TFL1 and BROTHEROF FT AND TFL1 clades. One gene in the TFL1 clade, PnTFL1, isexpressed in vegetative meristems, and transgenic Arabidopsisthat ectopically expressed PnTFL1 had a late-flowering phenotype.The expression patterns of two genes in the FT clade, PnFT1and PnFT2, suggested a role for them in the promotion of flowering,and transgenic Arabidopsis that ectopically expressed eitherPnFT1 or PnFT2 had an early-flowering phenotype.     

Genome-wide analysis of the chalcone synthase superfamily genes of <Emphasis Type="Italic">Physcomitrella patens</Emphasis>

Enzymes of the chalcone synthase (CHS) superfamily catalyze the production of a variety of secondary metabolites in bacteria, fungi and plants. Some of these metabolites have played important roles during the early evolution of land plants by providing protection from various environmental assaults including UV irradiation. The genome of the moss, Physcomitrella patens, contains at least 17 putative CHS superfamily genes. Three of these genes (PpCHS2b, PpCHS3 and PpCHS5) exist in multiple copies and all have corresponding ESTs. PpCHS11 and probably also PpCHS9 encode non-CHS enzymes, while PpCHS10 appears to be an ortholog of plant genes encoding anther-specific CHS-like enzymes. It was inferred from the genomic locations of genes comprising it that the moss CHS superfamily expanded through tandem and segmental duplication events. Inferred exon–intron architectures and results from phylogenetic analysis of representative CHS superfamily genes of P. patens and other plants showed that intron gain and loss occurred several times during evolution of this gene superfamily. A high proportion of P. patens CHS genes (7 of 14 genes for which the full sequence is known and probably 3 additional genes) are intronless, prompting speculation that CHS gene duplication via retrotransposition has occurred at least twice in the moss lineage. Analyses of sequence similarities, catalytic motifs and EST data indicated that a surprisingly large number (as many as 13) of the moss CHS superfamily genes probably encode active CHS. EST distribution data and different light responsiveness observed with selected genes provide evidence for their differential regulation. Observed diversity within the moss CHS superfamily and amenability to gene manipulation make Physcomitrella a highly suitable model system for studying expansion and functional diversification of the plant CHS superfamily of genes.     

Phylogenomic Analysis of the PEBP Gene Family in Cereals

The TFL1 and FT genes, which are key genes in the control of flowering time in Arabidopsis thaliana, belong to a small multigene family characterized by a specific phosphatidylethanolamine-binding protein domain, termed the PEBP gene family. Several PEBP genes are found in dicots and monocots, and act on the control of flowering time. We investigated the evolution of the PEBP gene family in cereals. First, taking advantage of the complete rice genome sequence and EST databases, we found 19 PEBP genes in this species, 6 of which were not previously described. Ten genes correspond to five pairs of paralogs mapped on known duplicated regions of the rice genome. Phylogenetic analysis of Arabidopsis and rice genes indicates that the PEBP gene family consists of three main homology classes (the so-called TFL1-LIKE, MFT-LIKE, and FT-LIKE subfamilies), in which gene duplication and/or loss occurred independently in Arabidopsis and rice. Second, phylogenetic analyses of genomic and EST sequences from five cereal species indicate that the three subfamilies of PEBP genes have been conserved in cereals. The tree structure suggests that the ancestral grass genome had at least two MFT-like genes, two TFL1-like genes, and eight FT-like genes. A phylogenomic approach leads to some hypotheses about conservation of gene function within the subfamilies. [Reviewing Editor: Dr. Yves Van de Peer]     

Functional Evolution of Phosphatidylethanolamine Binding Proteins in Soybean and Arabidopsis

Gene duplication provides resources for novel gene functions. Identification of the amino acids responsible for functional conservation and divergence of duplicated genes will strengthen our understanding of their evolutionary course. Here, we conducted a systemic functional investigation of phosphatidylethanolamine binding proteins (PEBPs) in soybean (Glycine max) and Arabidopsis thaliana. Our results demonstrated that after the ancestral duplication, the lineage of the common ancestor of the FLOWERING LOCUS T (FT) and TERMINAL FLOWER1 (TFL1) subfamilies functionally diverged from the MOTHER OF FT AND TFL1 (MFT) subfamily to activate flowering and repress flowering, respectively. They also underwent further specialization after subsequent duplications. Although the functional divergence increased with duplication age, we observed rapid functional divergence for a few pairs of young duplicates in soybean. Association analysis between amino acids and functional variations identified critical amino acid residues that led to functional differences in PEBP members. Using transgenic analysis, we validated a subset of these differences. We report clear experimental evidence for the functional evolution of the PEBPs in the MFT, FT, and TFL1 subfamilies, which predate the origin of angiosperms. Our results highlight the role of amino acid divergence in driving evolutionary novelty after duplication.     

Biological implications of the occurrence of 32 members of the XTH (xyloglucan endotransglucosylase/hydrolase) family of proteins in the bryophyte Physcomitrella patens

This comprehensive overview of the xyloglucan endotransglucosylase/hydrolase (XTH) family of genes and proteins in bryophytes, based on research using genomic resources that are newly available for the moss Physcomitrella patens, provides new insights into plant evolution. In angiosperms, the XTH genes are found in large multi‐gene families, probably reflecting the diverse roles of individual XTHs in various cell types. As there are fewer cell types in P. patens than in angiosperms such as Arabidopsis and rice, it is tempting to deduce that there are fewer XTH family genes in bryophytes. However, the present study unexpectedly identified as many as 32 genes that potentially encode XTH family proteins in the genome of P. patens, constituting a fairly large multi‐gene family that is comparable in size with those of Arabidopsis and rice. In situ localization of xyloglucan endotransglucosylase activity in this moss indicates that some P. patens XTH proteins exhibit biochemical functions similar to those found in angiosperms, and that their expression profiles are tissue‐dependent. However, comparison of structural features of families of XTH genes between P. patens and angiosperms demonstrated the existence of several bryophyte‐specific XTH genes with distinct structural and functional features that are not found in angiosperms. These bryophyte‐specific XTH genes might have evolved to meet morphological and functional needs specific to the bryophyte. These findings raise interesting questions about the biological implications of the XTH family of proteins in non‐seed plants.     

小立碗藓扩展蛋白基因家族的鉴定与生物信息学分析

扩展蛋白(Expansins,EXP)是一类基因家族,几乎参与了植物发育的全过程,从种子萌发到果实成熟都有扩展蛋白的参与。该研究利用生物信息学的方法对小立碗藓(Physcomitrella patens) Expansin基因家族成员进行鉴定,分析了其基因结构、染色体定位以及系统发生关系。结果表明:小立碗藓基因组中含有Expansin A(EXPA) 32个、Expansin-like A(EXLA) 6个,并未发现Expansin-like B(EXLB)及Expansin B(EXPB)。扩展蛋白氨基酸序列长度在228～290 aa之间,编码蛋白质具有两个保守的结构域Pollen_allerg_1和DPBB_1。蛋白质亚细胞定位预测结果表明:运用CELLO在线工具预测发现小立碗藓中约4/5的EXP家族基因定位于细胞外;而Euk-mPLoc预测结果则显示小立碗藓EXP基因家族成员全定位于细胞外。基因结构分析表明,小立碗藓中约68%Expansin基因有含有1～3个内含子。以上结果可为深入研究小立碗藓扩展蛋白基因的分子进化与生物学功能奠定基础。