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.     

ZCN8 encodes a potential orthologue of Arabidopsis FT florigen that integrates both endogenous and photoperiod flowering signals in maize

Higher plants use multiple perceptive measures to coordinate flowering time with environmental and endogenous cues. Physiological studies show that florigen is a mobile factor that transmits floral inductive signals from the leaf to the shoot apex. Arabidopsis FT protein is widely regarded as the archetype florigen found in diverse plant species, particularly in plants that use inductive photoperiods to flower. Recently, a large family of FT homologues in maize, the Zea CENTRORADIALIS (ZCN) genes, was described, suggesting that maize also contains FT-related proteins that act as a florigen. The product of one member of this large family, ZCN8, has several attributes that make it a good candidate as a maize florigen. Mechanisms underlying the floral transition in maize are less well understood than those of other species, partly because flowering in temperate maize is dependent largely on endogenous signals. The maize indeterminate1 (id1) gene is an important regulator of maize autonomous flowering that acts in leaves to mediate the transmission or production of florigenic signals. This study finds that id1 acts upstream of ZCN8 to control its expression, suggesting a possible new link to flowering in day-neutral maize. Moreover, in teosinte, a tropical progenitor of maize that requires short-day photoperiods to induce flowering, ZCN8 is highly up-regulated in leaves under inductive photoperiods. Finally, vascular-specific expression of ZCN8 in Arabidopsis complements the ft-1 mutation, demonstrating that leaf-specific expression of ZCN8 can induce flowering. These results suggest that ZCN8 may encode a florigen that integrates both endogenous and environmental signals in maize.     

The alleles at the E1 locus impact the expression pattern of two soybean FT-like genes shown to induce flowering in Arabidopsis

A small gene family of phosphatidyl ethanolamine-binding proteins (PEBP) has been shown to function as key regulators in flowering; in Arabidopsis thaliana the FT protein promotes flowering whilst the closely related TFL1 protein represses flowering. Control of flowering time in soybean [Glycine max (L.) Merrill] is important for geographic adaptation and maximizing yield. Soybean breeders have identified a series of loci, the E-genes, that control photoperiod-mediated flowering time, yet how these loci control flowering is poorly understood. The objectives of this study were to evaluate the expression of GmFT-like genes in the E1 near-isogenic line (NIL) background. Of the 20 closely related PEBP proteins in the soybean genome, ten are similar to the Arabidopsis FT protein. Expression analysis of these ten GmFT-like genes confirmed that only two are detectable in the conditions tested. Further analysis of these two genes in the E1 NILs grown under short-day (SD) and long-day (LD) conditions showed a diurnal expression and tissue specificity expression commensurate with soybean flowering time under SD and LD conditions, suggesting that these were good candidates for flowering induction in soybean. Arabidopsis ft mutant lines flowered early when transformed with the two soybean genes, suggesting that the soybean genes can complement the Arabidopsis FT function. Flowering time in E1 NILs is consistent with the differential expression of the two GmFT-like genes under SD and LD conditions, suggesting that the E1 locus, at least in part, impacts time to flowering through the regulation of soybean FT expression.     

The role of teosinte glume architecture (tga1) in coordinated regulation and evolution of grass glumes and inflorescence axes

? Hardened floral bracts and modifications to the inflorescence axis of grasses have been hypothesized to protect seeds from predation and/or aid seed dispersal, and have evolved multiple times independently within the family. Previous studies have demonstrated that mutations in the maize (Zea mays ssp. mays) gene teosinte glume architecture (tga1) underlie a reduction in hardened structures, yielding free fruits that are easy to harvest. It remains unclear whether the causative mutation(s) occurred in the cis-regulatory or protein-coding regions of tga1, and whether similar mutations in TGA1-like genes can explain variation in the dispersal unit in related grasses. ? To address these questions TGA1-like genes were cloned and sequenced from a number of grasses and analyzed phylogenetically in relation to morphology; protein expression was investigated by immunolocalization. ? TGA1-like proteins were expressed throughout the spikelet in the early development of all grasses, and throughout the flower of the grass relative Joinvillea. Later in development, expression patterns differed between Tripsacum dactyloides, maize and teosinte (Z. mays ssp. parviglumis). ? These results suggest an ancestral role for TGA1-like genes in early spikelet development, but do not support the hypothesis that TGA1-like genes have been repeatedly modified to affect glume and inflorescence axis diversification.     

Small auxin upregulated RNA (SAUR) gene family in maize: Identification,evolution, and its phylogenetic comparison with Arabidopsis,rice, and sorghum

Small auxin-up RNAs(SAURs)are the early auxin-responsive genes represented by a large multigene family in plants.Here,we identified 79 SAUR gene family members from maize(Zea mays subsp.mays)by a reiterative database search and manual annotation.Phylogenetic analysis indicated that the SAUR proteins from Arabidopsis,rice,sorghum,and maize had divided into 16 groups.These genes were non-randomly distributed across the maize chromosomes,and segmental duplication and tandem duplication contributed to the expansion of the maize SAUR gene family.Synteny analysis established orthology relationships and functional linkages between SAUR genes in maize and sorghum genomes.We also found that the auxin-responsive elements were conserved in the upstream sequences of maize SAUR members.Selection analyses identified some significant site-specific constraints acted on most SAUR paralogs.Expression profiles based on microarray data have provided insights into the possible functional divergence among members of the SAUR gene family.Quantitative real-time PCR analysis indicated that some of the 10 randomly selected ZmSAUR genes could be induced at least in maize shoot or root tissue tested.The results reveal a comprehensive overview of the maize SAUR gene family and may pave the way for deciphering their function during plant development.     

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]     

Small auxin upregulated RNA （SAUR） gene family in maize： Identification,evolution, and its phylogenetic comparison with Arabidopsis,rice, and sorghum

Small auxin-up RNAs （.SAURs） are the early auxin- responsive genes represented by a large multigene family in plants. Here, we identified 79 SAUR gene family members from maize （Zea mays subsp, mays） by a reiterative database search and manual annotation. Phylogenetic analysis indicated that the SAUR proteins from Arabidopsis, rice, sorghum, and maize had divided into 16 groups. These genes were non-randomly distributed across the maize chromosomes, and segmental duplication and tandem duplication contributed to the expansion of the maize .SAUR gene family. Synteny analysis established ortholos~J relationships and functional linkages between SAUR genes in maize and sorghum genomes. We also found that the auxin-responsive elements were conserved in the upstream sequences of maize SAUR members. Selection analyses identified some significant site-specific constraints acted on most SAUR paralogs. Expression profiles based on microarray data have provided insights into the possible functional divergence among members of the .SAUR gene family. Quantitative real-time PCR analysis indicated that some of the 10 randomly selected ZmSAUR genes could be induced at least in maize shoot or root tissue tested. The results reveal a comprehensive overview of the maize .SAUR gene family and may pave the way for deciphering their function during pJant development.     

Proteins from the FLOWERING LOCUS T‐like subclade of the PEBP family act antagonistically to regulate floral initiation in tobacco

Flowering is an important agronomic trait that often depends on the integration of photoperiod, vernalization, gibberellin and/or autonomous signaling pathways by regulatory proteins such as FLOWERING LOCUS T (FT), a member of the phosphatidylethanolamine‐binding protein (PEBP) family. Six PEBP family proteins control flowering in the model plant Arabidopsis thaliana, and their regulatory functions are well established, but variation in the number and structural diversity of PEBPs in different species means their precise functions must be determined on a case‐by‐case basis. We isolated four novel FT‐like genes from Nicotiana tabacum (tobacco), and determined their expression profiles in wild‐type plants and their overexpression phenotypes in transgenic plants. We found that all four genes were expressed in leaves under short‐day conditions, and at least NtFT3 expression was restricted to phloem companion cells. We also found that the NtFT1, NtFT2 and NtFT3 proteins are floral inhibitors (atypical for FT‐like proteins), whereas only NtFT4 is a floral inducer. We were unable to detect the expression of these genes under long‐day conditions, suggesting that all four tobacco FT‐like proteins may control flowering in response to short days. Phylogenetic analysis of PEBP family proteins and their functions in different solanaceous species confirmed that gene duplication and divergence within the FT‐like clade has led to the evolution of antagonistic regulators that may help to fine‐tune floral initiation in response to environmental cues.     

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.     

Expression and Functional Analysis of the ZCN1(ZmTFL1) Gene, a TERMINAL FLOWER 1 Homologue that Regulates the Vegetative to Reproductive Transition in Maize

TERMINAL FLOWER 1 (TFL1) homologs play critical roles in regulating flowering time and/or maintaining flowering of meristems. In this study, the gene of maize TFL1 ortholog ZmTFL1 (ZCN1) was cloned from both the tropical inbred line CML288 and temperate inbred line Huangzao 4, and the function of ZmTFL1 (ZCN1) was determined during different periods of floral development. Spatial and temporal expression patterns revealed that ZCN1 was predominantly localized in shoot apical meristems that develop into flowers, and only at low levels in leaves. To further identify the role of ZCN1 in floral development of maize, the morphology of shoot apices in maize during floral development was investigated using laser scanning confocal microscopy. Moreover, the relative levels of expression of ZCN1, ZCN8, DLF1, and ZAP1 genes were determined. Over-expression of ZCN1 partially complemented the late flowering phenotype in the tfl1-14 Arabidopsis mutant. Moreover, transgenic Arabidopsis plants exhibited indeterminate inflorescence with increased shoot length and higher numbers of trichomes on leaves. In addition, expression levels of AP1 were significantly down-regulated in 35S::ZCN1 transgenic Arabidopsis plants. These results indicated that ZCN1 as well as its homolog TFL1 in Arabidopsis are involved in the regulation of floral transition in maize.     

Genome-wide analysis of WOX gene family in rice, sorghum, maize, Arabidopsis and poplar

WUSCHEL-related homeobox(WOX)genes form a large gene family specifically expressed in plants.They are known to play important roles in regulating the development of plant tissues and organs by determining cell fate.Recent available whole genome sequences allow us to do more comprehensive phylogenetic analysis of the WOX genes in plants.In the present study,we identified 11 and 21 WOXs from sorghum(Sorghum bicolor)and maize(Zea mays),respectively.The 72 WOX genes from rice(Oryza sativa),sorghum,maize,Arabidopsis(Arabidopsis thaliana)and poplar(Populus trichocarpa)were grouped into three well supported clades with nine subgroups according to the amino acid sequences of their homodomains.Their phylogenetic relationship was also supported by the observation of the motifs outside the homodomain.We observed the variation of duplication events among the nine sub-groups between monocots and eudicots,for instance,more gene duplication events of WOXs within subgroup A for monocots,while,less for dicots in this subgroup.Furthermore,we observed the conserved intron/exon structural patterns of WOX genes in rice,sorghum and Arabidopsis.In addition,WUS(Wuschel)-box and EAR(the ERF-associated amphiphilic repression)-like motif were observed to be conserved among several WOX subgroups in these five plants.Comparative analysis of expression patterns of WOX genes in rice and Arabidopsis suggest that the WOX genes play conserved and various roles in plants.This work provides insights into the evolution of the WOX gene family and is useful for future research.     

Acceleration of flowering by overexpression of MFT (MOTHER OF FT AND TFL1)

MFT (MOTHER OF FT AND TFL1) is a member of a gene family that includes two important regulators, FT (FLOWERING LOCUS T) and TFL1 (TERMINAL FLOWER 1), in determination of flowering time in Arabidopsis. Although the functions of FT and TFL1 are assigned in the family, the roles of other members are largely unknown. Especially the sequence of MFT is homologous to both FT and TFL1, which act as a floral promoter and an inhibitor, respectively, making it difficult to predict the role of MFT. We performed genetic analyses of MFT to understand its role in floral development. Constitutive expression of MFT led to slightly early flowering under long days. However, a T-DNA insertion allele of MFT did not show obvious phenotype. Further genetic analyses with the loss-of-function alleles of FT, TFL1, and ATC (Arabidopsis Thaliana CENTRORADIALIS homologue) showed that a decrease of MFT activity did not enhance the phenotypes of the single mutants. Taken together, we suggest that MFT functions as a floral inducer and that it may act redundantly in determination of flowering time in Arabidopsis.     

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.     

The Arabidopsis heterochromatin protein1 homolog (TERMINAL FLOWER2) silences genes within the euchromatic region but not genes positioned in heterochromatin

TERMINAL FLOWER2 (TFL2) is the only homolog of heterochromatin protein1 (HP1) in the Arabidopsis genome. Because proteins of the HP1 family in fission yeast and animals act as key components of gene silencing in heterochromatin by binding to histone H3 methylated on lysine 9 (K9), here we examined whether TFL2 has a similar role in Arabidopsis. Unexpectedly, genes positioned in heterochromatin were not activated in tfl2 mutants. Moreover, the TFL2 protein localized preferentially to euchromatic regions and not to heterochromatic chromocenters, where K9-methylated histone H3 is clustered. Instead, TFL2 acts as a repressor of genes related to plant development, i.e. flowering, floral organ identity, meiosis and seed maturation. Up-regulation of the floral homeotic genes PISTILLATA, APETALA3, AGAMOUS and SEPALLATA3 in tfl2 mutants was independent of LEAFY or APETALA3, known activators of the above genes. In addition, transduced APETALA3 promoter fragments as short as 500 bp were sufficient for TFL2-mediated gene repression. Taken together, TFL2 silences specific genes within euchromatin but not genes positioned in heterochromatin of Arabidopsis.     

Arabidopsis TERMINAL FLOWER 2 gene encodes a heterochromatin protein 1 homolog and represses both FLOWERING LOCUS T to regulate flowering time and several floral homeotic genes

Floral transition should be strictly regulated because it is one of the most critical developmental processes in plants. Arabidopsis terminal flower 2 (tfl2) mutants show an early-flowering phenotype that is relatively insensitive to photoperiod, as well as several other pleiotropic phenotypes. We found that the early flowering of tfl2 is caused mainly by ectopic expression of the FLOWERING LOCUS T (FT) gene, a floral pathway integrator. Molecular cloning of TFL2 showed that it encodes a protein with homology to heterochromatin protein 1 (HP1) of animals and Swi6 of fission yeast. TFL2 protein localizes in subnuclear foci and expression of the TFL2 gene complemented yeast swi6(-) mutants. These results suggested that TFL2 might function as an HP1 in Arabidopsis: Gene expression analyses using DNA microarrays, however, did not show an increase in the expression of heterochromatin genes in tfl2 mutants but instead showed the upregulation of the floral homeotic genes APETALA3, PISTILLATA, AGAMOUS and SEPALLATA3. The pleiotropic phenotype of the tfl2 mutant could reflect the fact that TFL2 represses the expression of multiple genes. Our results demonstrate that despite its homology to HP1, TFL2 is involved in the repression of specific euchromatin genes and not heterochromatin genes in Arabidopsis.     

Whole-genome comparison of leucine-rich repeat extensins in Arabidopsis and rice. A conserved family of cell wall proteins form a vegetative and a reproductive clade

We have searched the Arabidopsis and rice (Oryza sativa) genomes for homologs of LRX1, an Arabidopsis gene encoding a novel type of cell wall protein containing a leucine-rich repeat (LRR) and an extensin domain. Eleven and eight LRX (LRR/EXTENSIN) genes have been identified in these two plant species, respectively. The LRX gene family encodes proteins characterized by a short N-terminal domain, a domain with 10 LRRs, a cysteine-rich motif, and a variable C-terminal extensin-like domain. Phylogenetic analysis performed on the conserved domains indicates the existence of two major clades of LRX proteins that arose before the eudicot/monocot divergence and then diversified independently in each lineage. In Arabidopsis, gene expression studies by northern hybridization and promoter::uidA fusions showed that the two phylogenetic clades represent a specialization into "reproductive" and "vegetative" LRXs. The four Arabidopsis genes of the "reproductive" clade are specifically expressed in pollen, whereas the seven "vegetative" genes are predominantly expressed in various sporophytic tissues. This separation into two expression classes is also supported by previous studies on maize (Zea mays) and tomato (Lycopersicon esculentum) LRX homologs and by information on available rice ESTs. The strong conservation of the amino acids responsible for the putative recognition specificity of the LRR domain throughout the family suggests that the LRX proteins interact with similar ligands.