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.     

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.     

DFL, a FLORICAULA/LEAFY homologue gene from Dendranthema lavandulifolium is expressed both in the vegetative and reproductive tissues

FLO/LFY homologue genes were initially characterized as floral meristem identity genes and play a key role in flower development among diverse species. The inflorescence organization of chrysanthemum differs from typical dicotyledons such as Arabidopsis and Antirrhinum as clear sepals are absent, and instead, a pappus, a rudimentary sepal, is formed. To understand the mechanism of reproduction of chrysanthemum at the molecular level, DFL, a FLORICAULA/LEAFY homologous gene, was cloned from Dendranthema lavandulifolium, which is one of the original species of chrysanthemum. The DFL gene consists of a 1,236-bp open reading frame and encodes a putative protein of 412 amino acids, which is 63% identical to LFY and 70% to FLO. The expression patterns of DFL during the flower development were analyzed, and RT-PCR results showed that DFL was strongly expressed in the flower bud. In situ hybridization experiments showed that it is strongly expressed in the inflorescence bract, petal and stamen primordial tissues throughout the inflorescence development. Its expression signals were also detected in stems, leaf primordial tissues and developing inflorescence bracts.     

CnFL,a FLORICAULA/LEAFY homolog in Chrysanthemum nankingense is dramatically upregulated in induced shoot apical meristems

A FLORICOULA/LEAFY (FLO/LFY) homolog CnFL gene was isolated from the flower bud of a short-day Chrysanthemum nankingense plant during the flowering induction period. The sequence of CnFL contained a 1236 bp open reading frame that encoded a putative protein of 412 amino acids, which shared 68.67% homology with FLO and 60.23% homology with LFY. The spatial expression patterns of CnFL were analyzed using quantitative real-time PCR in different tissues and the apical meristem during short-day flowering induction. The results indicated that CnFL was highly expressed in the flower buds while its expression was also detected in the stems, young leaves, and vegetative apical meristem. During the period of flowering induction, CnFL expression increased remarkably and reached its highest levels after 15 days of induction. The expression of CnFL in the apical shoot after short-day flowering induction indicates that CnFL regulation is controlled primarily by photoperiodicity.     

Apple has two orthologues of FLORICAULA/LEAFY involved in flowering

Two orthologues of FLORICAULA/LEAFY, AFL1 and AFL2 (apple FLO/LFY), were isolated from the floral buds of apple trees. Their expression was detected in various tissues and during differentiation of the floral buds. Furthermore, the flowering effectiveness of each gene was assessed with transgenic Arabidopsis. Both AFL1 and AFL2 showed high homology to each other (90%) and a high degree of similarity to PTLF and PEAFLO (70%), which are homologues of FLO/LFY from poplar and pea, respectively. RNA blot analysis showed that AFL1 was expressed only in the floral bud during the transition from vegetative to reproductive growth, whereas AFL2 was expressed in vegetative shoot apex, floral buds, floral organs and root. Genomic Southern analysis showed that apple had other homologues in addition to AFL1 and AFL2. The transgenic Arabidopsis with over-expressed AFL2 showed accelerated flowering and gave rise to several solitary flowers from rosette axils directly. AFL1 had similar effects, but the phenotypes of the transgenic Arabidopsis with AFL1 were weaker than those with AFL2. These results suggest that both genes are involved in flower differentiation in apple.     

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.     

Molecular evolution of FLORICAULA/LEAFY orthologs in the Andropogoneae (Poaceae)

Members of the grass family (Poaceae) exhibit a broad range of inflorescence structures and other morphologies, making the grasses an interesting model system for studying the evolution of development. Here we present an analysis of the molecular evolution of FLORICAULA/LEAFY-like genes, which are important developmental regulatory loci known to affect inflorescence development in a wide range of flowering plant species. We have focused on sequences from the Andropogoneae, a tribe within the grass family that includes maize (Zea mays ssp. mays) and Sorghum (Sorghum bicolor). The FLORICAULA/LEAFY gene phylogeny we generated largely agrees with previously published phylogenies for the Andropogoneae using other nuclear genes but is unique in that it includes both members of one of the many duplicate gene sets present in maize. The placement of these sequences in the phylogeny suggests that the duplication of the maize FLORICAULA/LEAFY orthologs, zfl1 and zfl2, is a consequence of a proposed tetraploidy event that occurred in the common ancestor of Zea and a closely related genus, Tripsacum. Our data are consistent with the hypothesis that the transcribed regions of the FLORICAULA/LEAFY-like genes in the Andropogoneae are functionally constrained at both nonsynonymous and synonymous sites and show no evidence of directional selection. We also examined conservation of short noncoding sequences in the first intron, which may play a role in gene regulation. Finally, we investigated the genetic diversity of one of the two maize FLORICAULA/LEAFY orthologs, zfl2, in maize and its wild ancestor, teosinte (Z. mays ssp. parviglumis), and found no evidence for selection pressure resulting from maize domestication within the zfl2-coding region.     

The rubber tree (Hevea brasiliensis Muell. Arg.) homologue of the LEAFY/FLORICAULA gene is preferentially expressed in both male and female floral meristems

The rubber tree (Hevea brasiliensis Muell. Arg.) is an important source of natural rubber in tropical regions and, as with many woody species, shows a long juvenile phase. To understand the genetic and molecular mechanisms underlying the reproductive process in rubber trees, H. brasiliensis RRIM600 flower and inflorescence development have been characterized, the rubber tree FLORICAULA/LEAFY (FLO/LFY) orthologue, HbLFY, cloned, and its expression patterns were analysed during vegetative and reproductive development. The rubber tree, similar to other Euphorbiaceae species, produces lateral inflorescences containing male, female, and bisexual flowers. HbLFY is expressed in lateral meristems that give rise to inflorescences and in all flower meristems, consistent with a role in reproductive development. Complementation studies using Arabidopsis lfy mutants indicated that the biological function of LFY might be conserved among Brassicaceae and Euphorbiaceae species.     

Potential phylogenetic utility of the nuclear FLORICAULA/LEAFY second intron: comparison with three chloroplast DNA regions in Amorphophallus (Araceae)

FLORICAULA/LEAFY (FLO/LFY) is a single-copy nuclear-encoded homeotic gene containing two introns. We have investigated the utility of the second intron of FLO/LFY (FLint2) as a tool for phylogeny reconstruction at lower taxonomic levels. As an example, the phylogeny of 46 Amorphophallus, two Pseudodracontium, and four outgroup species is reconstructed using maximum parsimony and maximum likelihood analyses of FLint2 sequences. We designed new primers based on conserved sequences of the second and third exon for use in a range of Aroid taxa to amplify and sequence the second intron. In Amorphophallus FLint2 proved to be rather short (143-222 bp), highly variable and unsaturated. In all but two species a single amplification product was found. Results from phylogenetic analysis of FLint2 are largely congruent with results using the chloroplast regions rbcL, matK, and trnL, and compare favorably in percentage of informative characters, overall homoplasy levels, number of well-supported clades in consensus trees and resolution of ingroup relationships within Amorphophallus. When amplification products are not too large, alignment is relatively straightforward, and sequences are used in combination with other fast evolving markers, the FLint2 intron may be a valuable new tool for phylogenetic studies at lower taxonomic levels.     

Diverse effects of overexpression of LEAFY and PTLF, a poplar (Populus) homolog of LEAFY/FLORICAULA, in transgenic poplar and Arabidopsis

PTLF, the Populus trichocarpa homolog of LEAFY (LFY) and FLORICAULA, was cloned to assess its function in a dioecious tree species. In situ hybridization studies showed that the gene was expressed most strongly in developing inflorescences. Expression was also seen in leaf primordia and very young leaves, most notably in apical vegetative buds near inflorescences, but also in seedlings. Although ectopic expression of the PTLF cDNA in Arabidopsis accelerated flowering, only one of the many tested transgenic lines of Populus flowered precociously. The majority of trees within a population of 3-year-old transgenic hybrid Populus lines with PTLF constitutively expressed showed few differences when compared to controls. However, phenotypic effects on growth rate and crown development, but not flowering, were seen in some trees with strong PTLF expression and became manifest only as the trees aged. Competence to respond to overexpression of LFY varied widely among Populus genotypes, giving consistent early flowering in only a single male P. tremula x P. tremuloides hybrid and causing gender change in another hybrid genotype. PTLF activity appears to be subject to regulation that does not affect heterologously expressed LFY, and is dependent upon tree maturation. Both genes provide tools for probing the mechanisms of delayed competence to flower in woody plants.     

Identification and Characterization of PpLFL, a Homolog of FLORICAULA/LEAFY in Peach (Prunus persica)

A LEAFY/FLORICAULA (LFY/FLO) homolog PpLFL (P runus p ersica L EAFY/ F LORICAULA L ike) gene was isolated from axillary buds of peach (Prunus persica (L.) Batsch. cv. Bayuecui) during flower induction period. The open reading frame of PpLFL spanned 1,248 bp, encoding a putative protein of 415 amino acid residues, which was with high similarity (50.48 %–84.69 %) to other FLO/LFY inferred proteins from different species. The spatial expression patterns of PpLFL were detected in axillary buds during the periods of flower induction by using immunohistolocalisation. The results showed that PpLFL gene was mainly expressed during flower induction time, and also in leaf and petal promordia at the SAM. For further functional analysis, the PpLFL was constitutively expressed in the Arabidopsis lfy mutant background, and the results showed that overexpression of PpLFL under the control of CaMV 35S promoter can accelerate flowering and give rise to normal flower organs. Our results suggest that PpLFL might play an important role in flower induction, and could act as a functional flower meristem identity gene in peach.     

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.     

Pleiotropic effects of the duplicate maize FLORICAULA/LEAFY genes zfl1 and zfl2 on traits under selection during maize domestication

Phenotypic variation on which selection can act during evolution may be caused by variation in activity level of developmental regulatory genes. In many cases, however, such genes affect multiple traits. This situation can lead to co-evolution of traits, or evolutionary constraint if some pleiotropic effects are detrimental. Here, we present an analysis of quantitative traits associated with gene copy number of two important maize regulatory genes, the duplicate FLORICAULA/LEAFY orthologs zfl1 and zfl2. We found statistically significant associations between several quantitative traits and copy number of both zfl genes in several maize genetic backgrounds. Despite overlap in traits associated with these duplicate genes, zfl1 showed stronger associations with flowering time, while zfl2 associated more strongly with branching and inflorescence structure traits, suggesting some divergence of function. Since zfl2 associates with quantitative variation for ear rank and also maps near a quantitative trait locus (QTL) on chromosome 2 controlling ear rank differences between maize and teosinte, we tested whether zfl2 might have been involved in the evolution of this trait using a QTL complementation test. The results suggest that zfl2 activity is important for the QTL effect, supporting zfl2 as a candidate gene for a role in morphological evolution of maize.     

Arabidopsis SKP1, a homologue of a cell cycle regulator gene, is predominantly expressed in meristematic cells

The yeast SKP1 gene and its human homolog p19 ^skp1 encode a kinetochore protein required for cell cycle progression at both the DNA synthesis and mitosis phases of the cell cycle. In orchids we identified a cDNA (O108) that is expressed in early stages of ovule development and is homologous to the yeast SKP1. Based on the orchid O108 cDNA clone, we identified and characterized an Arabidopsis thaliana (L.) Heynh. cDNA designated ATskp1 that also has high sequence similarity to yeast SKP1. The Arabidopsis ATskp1 is a single-copy gene that mapped to chromosome 1. The expression of the ATskp1 gene was highly correlated with meristem activity in that its mRNA accumulated in all of the plant meristems including the vegetative shoot meristem, inflorescence and floral meristems, root meristem, and in the leaf and floral organ primordia. In addition, ATskp1 was also highly expressed in the dividing cells of the developing embryo, and in other cells that become multinucleate or undergo endoreplication events such as the endosperm free nuclei, the tapetum and the endothelium. Based on its spatial pattern of expression, ATskp1 is a marker for cells undergoing division and may be required for meristem activity. Received: 6 June 1997 / Accepted: 2 July 1997     

Lox6, a leech Dfd ortholog, is expressed in the central nervous system and in peripheral sensory structures

 Sequence analysis of a newly isolated Hirudo medicinalis cDNA containing an Antennapedia (Antp)-class homeobox suggests that the corresponding gene, Lox6, is an ortholog of the Drosophila Deformed (Dfd) gene. In situ hybridization of whole-mounted preparations shows that the major sites of Lox6 expression during embryogenesis are the central nervous system (CNS) and the peripheral sensory system. Lox6 mRNA can be detected in a subset of neurons in each ganglion from the subesophageal ganglion (RG2) to the most posterior ganglion, with the highest level of expression seen in RG3. Peripherally, Lox6 is expressed principally in the primordia of the sensillae and in the eyes. This pattern of expression of Lox6 suggests that one of its functions may be to contribute to the diversification of neuronal phenotypes. Received: 16 August 1997/Accepted: 20 December 1997