Relaxed Selection Among Duplicate Floral Regulatory Genes in Lamiales

Polyploidization is a prevalent mode of genome diversification within plants. Most gene duplicates arising from polyploidization (paralogs) are typically lost, although a subset may be maintained under selection due to dosage, partitioning of gene function, or acquisition of novel functions. Because they experience selection in the presence of other duplicate loci across the genome, interactions among genes may also play a significant role in the maintenance of paralogs resulting from polyploidization. Previously, we identified duplicates of the genes LFY/FLO and AP3/DEF that directly interact in a floral regulatory pathway and are thought to be the result of ancient polyploidization in the Lamiales (> 50 mya). Although duplicates of MADS box genes including AP3/DEF are common throughout the angiosperm lineage, LFY/FLO duplicates in Lamiales are the first reported outside of tetraploid taxa. In order to explore hypotheses for the joint preservation of these interacting floral regulatory genes including novel LFY/FLO paralogs, here we clone FLO and DEF duplicates from additional Lamiales taxa and apply codon substitution models to test how selection acts on both genes following duplication. We find acceleration in the ratio of nonsynonymous-to-synonymous nucleotide substitutions for one (FLO) or both (DEF) paralogs that appears to be due to relaxed purifying selection as opposed to positive selection and shows a different pattern among functional domains of these genes. Several mechanisms are discussed that might be responsible for preservation of co-orthologs of FLO and DEF in Lamiales, including interactions among the genes of this regulatory pathway. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Yves Van de Peer]     

Flowering genes in Metrosideros fit a broad herbaceous model encompassing Arabidopsis and Antirrhinum

Molecular studies were conducted on Metrosideros excelsa to determine if the current genetic models for flowering with regard to inflorescence and floral meristem identity genes in annual plants were applicable to a woody perennial. MEL , MESAP1 and METFL1 , the fragments of LEAFY ( LFY ), APETALA1 ( AP1 ) and TERMINAL FLOWER1 ( TFL1 ) equivalents, respectively, were isolated from M. excelsa . Temporal expression patterns showed that MEL and MESAP1 exhibited a bimodal pattern of expression. Expression exhibited during early floral initiation in autumn was followed by down-regulation during winter, and up-regulation in spring as floral organogenesis occurred. Spatial expression patterns of MEL showed that it had greater similarity to FLORICAULA ( FLO ) than to LFY , whereas MESAP1 was more similar to AP1 than SQUAMOSA . The interaction between MEL and METFL1 was more similar to the interaction between FLO and CENTRORADIALIS than that between LFY and TFL1 . Consequently, the three genes from M. excelsa fit a broader herbaceous model encompassing Antirrhinum as well as Arabidopsis , but with differences, such as the bimodal pattern of expression seen with MEL and MESAP1 . In mid-winter, at the time when both MEL and MESAP1 were down-regulated, GA₁ was below the level of detection in M. excelsa buds. Even though application of gibberellin inhibits flowering in members of the Myrtaceae, MEL was responsive to gibberellin with expression in juvenile plants up-regulated by GA₃. However, MESAP1 was not up-regulated indicating that meristem competence was also probably required to promote flowering in M. excelsa .     

Control of compound leaf development by FLORICAULA/LEAFY ortholog SINGLE LEAFLET1 in Medicago truncatula

Molecular genetic studies suggest that FLORICAULA (FLO)/LEAFY (LFY) orthologs function to control compound leaf development in some legume species. However, loss-of-function mutations in the FLO/LFY orthologs result in reduction of leaf complexity to different degrees in Pisum sativum and Lotus japonicus. To further understand the role of FLO/LFY orthologs in compound leaf development in legumes, we studied compound leaf developmental processes and characterized a leaf development mutant, single leaflet1 (sgl1), from the model legume Medicago truncatula. The sgl1 mutants exhibited strong defects in compound leaf development; all adult leaves in sgl1 mutants are simple due to failure in initiating lateral leaflet primordia. In addition, the sgl1 mutants are also defective in floral development, producing inflorescence-like structures. Molecular cloning of SGL1 revealed that it encodes the M. truncatula FLO/LFY ortholog. When properly expressed, LFY rescued both floral and compound leaf defects of sgl1 mutants, indicating that LFY can functionally substitute SGL1 in compound leaf and floral organ development in M. truncatula. We show that SGL1 and LFY differed in their promoter activities. Although the SGL1 genomic sequence completely rescued floral defects of lfy mutants, it failed to alter the simple leaf structure of the Arabidopsis thaliana plants. Collectively, our data strongly suggest that initiation of lateral leaflet primordia required for compound leaf development involves regulatory processes mediated by the SGL1 function in M. truncatula.     

Topological incongruence between nuclear and chloroplast DNA trees suggesting hybridization in the urophyllum group of the genus Fagopyrum (Polygonaceae)

We performed phylogenetic analyses of Fagopyrum species in the urophyllum group based on nucleotide sequences of two nuclear genes, FLORICAULA/LEAFY (FLO/LFY) and AGAMOUS (AG), and three segments of chloroplast DNA (cpDNA), rbcL-accD, trnK intron, and trnC-rpoB spacer. The FLO/LFY and AG sequences turned out to be phylogenetically more informative at the intrageneric level than the cpDNA sequences. Congruence among these gene trees, inferred by a maximum-likelihood (ML) method, demonstrated that topologies were partially incongruent between the nuclear and chloroplast DNA phylogenies. The nuclear DNA sequence data supported a monophyletic relation of F. statice, F. gilesii, and F. jinshaense, whereas the former two species formed another monophyletic relation with the F. capillatum-F. gracilipes-F. gracilipedoides-F. rubifolium clade excluding F. jinshaense in the synthetic cpDNA phylogeny. In addition, two divergent sequences of FLO/LFY were found in F. rubifolium (tetraploid). One of these was sister to F. gracilipedoides and another was sister to F. statice, and a monophyletic relation of these two genes was rejected by a bootstrap analysis. These results suggest that hybridization may have occurred during diversification of Fagopyrum species in the urophyllum group, and that F. rubifolium is possibly allotetraploid species.     

FALSIFLORA, the tomato orthologue of FLORICAULA and LEAFY, controls flowering time and floral meristem identity

Characterization of the tomato falsiflora mutant shows that fa mutation mainly alters the development of the inflorescence resulting in the replacement of flowers by secondary shoots, but also produces a late-flowering phenotype with an increased number of leaves below first and successive inflorescences. This pattern suggests that the FALSIFLORA (FA) locus regulates both floral meristem identity and flowering time in tomato in a similar way to the floral identity genes FLORICAULA (FLO) of Antirrhinum and LEAFY (LFY) of Arabidopsis. To analyse whether the fa phenotype is the result of a mutation in the tomato FLO/LFY gene, we have cloned and analysed the tomato FLO/LFY homologue (TOFL) in both wild-type and fa plants following a candidate gene strategy. The wild-type gene is predicted to encode a protein sharing 90% identity with NFL1 and ALF, the FLO/LFY-like proteins in Nicotiana and Petunia, and about 80 and 70% identity with either FLO or LFY. In the fa mutant, however, the gene showed a 16 bp deletion that results in a frameshift mutation and in a truncated protein. The co-segregation of this deletion with the fa phenotype in a total of 240 F2 plants analysed supports the idea that FA is the tomato orthologue to FLO and LFY. The gene is expressed in both vegetative and floral meristems, in leaf primordia and leaves, and in the four floral organs. The function of this gene in comparison with other FLO/LFY orthologues is analysed in tomato, a plant with a sympodial growth habit and a cymose inflorescence development.     

The matrix coalescent and an application to human single-nucleotide polymorphisms

The floral developmental pathway in Arabidopsis thaliana is composed of several interacting regulatory genes, including the inflorescence architecture gene TERMINAL FLOWER1 (TFL1), the floral meristem identity genes LEAFY (LFY), APETALA1 (AP1), and CAULIFLOWER (CAL), and the floral organ identity genes APETALA3 (AP3) and PISTILLATA (PI). Molecular population genetic analyses of these different genes indicate that the coding regions of AP3 and PI, as well as AP1 and CAL, share similar levels and patterns of nucleotide diversity. In contrast, the coding regions of TFL1 and LFY display a significant reduction in nucleotide variation, suggesting that these sequences have been subjected to a recent adaptive sweep. Moreover, the promoter of TFL1, unlike its coding region, displays high levels of diversity organized into two distinct haplogroups that appear to be maintained by selection. These results suggest that patterns of molecular evolution differ among regulatory genes in this developmental pathway, with the earlier acting genes exhibiting evidence of adaptive evolution.     

Interactions among APETALA1, LEAFY, and TERMINAL FLOWER1 specify meristem fate.

Upon floral induction, the primary shoot meristem of an Arabidopsis plant begins to produce flower meristems rather than leaf primordia on its flanks. Assignment of floral fate to lateral meristems is primarily due to the cooperative activity of the flower meristem identity genes LEAFY (LFY), APETALA1 (AP1), and CAULIFLOWER. We present evidence here that AP1 expression in lateral meristems is activated by at least two independent pathways, one of which is regulated by LFY. In lfy mutants, the onset of AP1 expression is delayed, indicating that LFY is formally a positive regulator of AP1. We have found that AP1, in turn, can positively regulate LFY, because LFY is expressed prematurely in the converted floral meristems of plants constitutively expressing AP1. Shoot meristems maintain an identity distinct from that of flower meristems, in part through the action of genes such as TERMINAL FLOWER1 (TFL1), which bars AP1 and LFY expression from the influorescence shoot meristem. We show here that this negative regulation can be mutual because TFL1 expression is downregulated in plants constitutively expressing AP1. Therefore, the normally sharp phase transition between the production of leaves with associated shoots and formation of the flowers, which occurs upon floral induction, is promoted by positive feedback interactions between LFY and AP1, together with negative interactions of these two genes with TFL1.     

The duplicated B-class heterodimer model: whorl-specific effects and complex genetic interactions in Petunia hybrida flower development

In both Antirrhinum (Antirrhinum majus) and Arabidopsis (Arabidopsis thaliana), the floral B-function, which specifies petal and stamen development, is embedded in a heterodimer consisting of one DEFICIENS (DEF)/APETALA3 (AP3)-like and one GLOBOSA (GLO)/PISTILLATA (PI)-like MADS box protein. Here, we demonstrate that gene duplications in both the DEF/AP3 and GLO/PI lineages in Petunia hybrida (petunia) have led to a functional diversification of their respective members, which is reflected by partner specificity and whorl-specific functions among these proteins. Previously, it has been shown that mutations in PhDEF (formerly known as GREEN PETALS) only affect petal development. We have isolated insertion alleles for PhGLO1 (FLORAL BINDING PROTEIN1) and PhGLO2 (PETUNIA MADS BOX GENE2) and demonstrate unique and redundant properties of PhDEF, PhGLO1, and PhGLO2. Besides a full homeotic conversion of petals to sepals and of stamens to carpels as observed in phglo1 phglo2 and phdef phglo2 flowers, we found that gene dosage effects for several mutant combinations cause qualitative and quantitative changes in whorl 2 and 3 meristem fate, and we show that the PHDEF/PHGLO1 heterodimer controls the fusion of the stamen filaments with the petal tube. Nevertheless, when the activity of PhDEF, PhGLO1, and PhGLO2 are considered jointly, they basically appear to function as DEF/GLO does in Antirrhinum and to a lesser extent as AP3/PI in Arabidopsis. By contrast, our data suggest that the function of the fourth B-class MADS box member, the paleoAP3-type PETUNIA HYBRIDA TM6 (PhTM6) gene, differs significantly from the known euAP3-type DEF/AP3-like proteins; PhTM6 is mainly expressed in the developing stamens and ovary of wild-type flowers, whereas its expression level is upregulated in whorls 1 and 2 of an A-function floral mutant; PhTM6 is most likely not involved in petal development. The latter is consistent with the hypothesis that the evolutionary origin of the higher eudicot petal structure coincided with the appearance of the euAP3-type MADS box genes.     

Compound leaf development and evolution in the legumes

Across vascular plants, Class 1 KNOTTED1-like (KNOX1) genes appear to play a critical role in the development of compound leaves. An exception to this trend is found in the Fabaceae, where pea (Pisum sativum) uses UNIFOLIATA, an ortholog of the floral regulators FLORICAULA (FLO) and LEAFY (LFY), in place of KNOX1 genes to regulate compound leaf development. To assess the phylogenetic distribution of KNOX1-independent compound leaf development, a survey of KNOX1 protein expression across the Fabaceae was undertaken. The majority of compound-leafed Fabaceae have expression of KNOX1 proteins associated with developing compound leaves. However, in a large subclade of the Fabaceae, the inverted repeat-lacking clade (IRLC), of which pea is a member, KNOX1 expression is not associated with compound leaves. These data suggest that the FLO/LFY gene may function in place of KNOX1 genes in generating compound leaves throughout the IRLC. The contribution of FLO/LFY to leaf complexity in a member of the Fabaceae outside of the IRLC was examined by reducing expression of FLO/LFY orthologs in transgenic soybean (Glycine max). Transgenic plants with reduced FLO/LFY expression showed only slight reductions in leaflet number. Overexpression of a KNOX1 gene in alfalfa (Medicago sativa), a member of the IRLC, resulted in an increase in leaflet number. This implies that KNOX1 targets, which promote compound leaf development, are present in alfalfa and are still sensitive to KNOX1 regulation. These data suggest that KNOX1 genes and the FLO/LFY gene may have played partially overlapping roles in compound leaf development in ancestral Fabaceae but that the FLO/LFY gene took over this role in the IRLC.     

Organization of the chromosome region harboring a FLORICAULA/LEAFY gene in Liriodendron

FLORICAULA/LEAFY (FLO/LFY) plays an important role in the reproductive transition and controls flower spatial patterning by inducing the expression of the ABC floral organ identity genes. In this study, we sequenced two bacterial artificial chromosomes harboring a FLO/LFY and three other genes from yellow-poplar (Liriodendron tulipifera L.) and compared the gene order in this locus between several species. Besides the conserved terminal domains, key residues involved in interactions with DNA bases, backbone, and in dimerization were also conserved in the yellow-poplar FLO/LFY. Phylogenetic analysis of the FLO/LFY amino acid sequences placed yellow-poplar closer to eudicots than to monocotyledonous species. We found that gene content and order in this region of the yellow-poplar genome was more similar to corresponding regions in Vitis vinifera L., Carica papaya L., Populus trichocarpa Torr. & Gray, and Ricinus communis L., regardless of the evolutionary relationship. In addition, evidence for transposition, large insertions, and duplications were found, suggesting multiple and complex mechanisms of basal angiosperm genome evolution.     

Cloning and characterization of a FLORICAULA/LEAFY ortholog, PFL, in polygamous papaya

The homologous genes FLORICAULA （FLO） in Antirrhinum and LEAFY （LFY） in Arabidopsis are known to regulate the initiation of flowering in these two distantly related plant species. These genes are necessary also for the expression of downstream genes that control floral organ identity. We used Arabidopsis LFY cDNA as a probe to clone and sequence a papaya ortholog of LFY, PFL. It encodes a protein that shares 61% identity with the Arabidopsis LFY gene and 71% identity with the LFY homologs of the two woody tree species： California sycamore （Platanus racemosa） and black cottonwood （Populus trichocarpa）. Despite the high sequence similarity within two conserved regions, the N-terminal proline-rich motif in papaya PFL differs from other members in the family. This difference may not affect the gene function of papaya PFL, since an equally divergent but a functional LFY ortholog NEEDLY of Pinus radiata has been reported. Genomic and BAC Southern analyses indicated that there is only one copy of PFL in the papaya genome. In situ hybridization experiments demonstrated that PFL is expressed at a relatively low level in leaf primordia, but it is expressed at a high level in the floral meristem. Quantitative PCR analyses revealed that PFL was expressed in flower buds of all three sex types - male, female, and hermaphrodite with marginal difference between hermaphrodite and unisexual flowers. These data suggest that PFL may play a similar role as LFY in flower development and has limited effect on sex differentiation in papaya.     

Genetic Interactions That Regulate Inflorescence Development in Arabidopsis

In Arabidopsis, floral meristems arise in continuous succession directly on the flanks of the inflorescence meristem. Thus, the pathways that regulate inflorescence and floral meristem identity must operate both simultaneously and in close spatial proximity. The TERMINAL FLOWER 1 (TFL1) gene of Arabidopsis is required for normal inflorescence meristem function, and the LEAFY (LFY), APETALA 1 (AP1), and APETALA 2 (AP2) genes are required for normal floral meristem function. We present evidence that inflorescence meristem identity is promoted by TFL1 and that floral meristem identity is promoted by parallel developmental pathways, one defined by LFY and the other defined by AP1/AP2. Our analysis suggests that the acquisition of meristem identity during inflorescence development is mediated by antagonistic interactions between TFL1 and LFY and between TFL1 and AP1/AP2. Based on this study, we propose a simple model for the genetic regulation of inflorescence development in Arabidopsis. This model is discussed in relation to the proposed interactions between the inflorescence and the floral meristem identity genes and in regard to other genes that are likely to be part of the genetic hierarchy regulating the establishment and maintenance of inflorescence and floral meristems.     

Uncovering genetic and molecular interactions among floral meristem identity genes in Arabidopsis thaliana

The inflorescence meristem produces floral primordia that remain undifferentiated during the first stages of flower development. Genes controlling floral meristem identity include LEAFY (LFY), APETALA1 (AP1), CAULIFLOWER (CAL), LATE MERISTEM IDENTITY 1 (LMI1), SHORT VEGETATIVE PHASE (SVP) and AGAMOUS-LIKE24 (AGL24). The lfy mutant shows partial reversions of flowers into inflorescence shoot-like structures and this phenotype is enhanced in the lfy ap1 double mutant. Here we show that combining the lfy mutant with agl24 and svp single mutants or with the agl24 svp double mutant enhances the lfy phenotype and that the lfy agl24 svp triple mutant phenocopies the lfy ap1 double mutant. Analysis of the molecular interactions between LFY, AGL24 and SVP showed that LFY is a repressor of AGL24 and SVP, whereas LMI1 is a positive regulator of these genes. Moreover, AGL24 and SVP positively regulate AP1 and LFY by direct binding to their regulatory regions. Since all these genes are important for establishing floral meristem identity, regulatory loops are probably important to maintain the correct relative expression levels of these genes.     

The ASK1 gene regulates B function gene expression in cooperation with UFO and LEAFY in Arabidopsis

The Arabidopsis floral regulatory genes APETALA3 (AP3) and PISTILLATA (PI) are required for the B function according to the ABC model for floral organ identity. AP3 and PI expression are positively regulated by the LEAFY (LFY) and UNUSUAL FLORAL ORGANS (UFO) genes. UFO encodes an F-box protein, and we have shown previously that UFO genetically interacts with the ASK1 gene encoding a SKP1 homologue; both the F-box containing protein and SKP1 are subunits of ubiquitin ligases. We show here that the ask1-1 mutation can enhance the floral phenotypes of weak lfy and ap3 mutants; therefore, like UFO, ASK1 also interacts with LFY and AP3 genetically. Furthermore, our results from RNA in situ hybridizations indicate that ASK1 regulates early AP3 and PI expression. These results support the idea that UFO and ASK1 together positively regulate AP3 and PI expression. We propose that the UFO and ASK1 proteins are components of a ubiquitin ligase that mediates the proteolysis of a repressor of AP3 and PI expression. Our genetic studies also indicate that ASK1 and UFO play a role in regulating the number of floral organ primordia, and we discuss possible mechanisms for such a regulation.     

Regulation of SUP expression identifies multiple regulators involved in arabidopsis floral meristem development

During the course of flower development, floral homeotic genes are expressed in defined concentric regions of floral meristems called whorls. The SUPERMAN (SUP, also called FLO10) gene, which encodes a C2H2-type zinc finger protein, is involved in maintenance of the stamen/carpel whorl boundary (the boundary between whorl 3 and whorl 4) in Arabidopsis. Here, we show that the regulation of SUP expression in floral meristems is complex, consisting of two distinct phases, initiation and maintenance. The floral meristem identity gene LEAFY (LFY) plays a role in the initiation phase through at least two pathways, which differ from each other in the involvement of two homeotic genes, APETALA3 (AP3) and PISTILLATA (PI). AP3, PI, and another homeotic gene, AGAMOUS (AG), are further required for SUP expression in the later maintenance phase. Aside from these genes, there are other as yet unidentified genes that control both the temporal and spatial patterns of SUP expression in whorl 3 floral meristems. SUP appears to act transiently, probably functioning to trigger a genetic circuit that creates the correct position of the whorl 3/whorl 4 boundary.