LFY Homologs Research Articles

Roles and evolution of four LEAFY homologs in floral patterning and leaf development in woodland strawberry.

The plant-specific transcription factor LEAFY (LFY), generally maintained as a single-copy gene in most angiosperm species, plays critical roles in flower development. The woodland strawberry (Fragaria vesca) possesses four LFY homologs in the genome; however, their respective functions and evolution remain unknown. Here, we identified and validated that mutations in one of the four LFY homologs, FveLFYa, cause homeotic conversion of floral organs and reiterative outgrowth of ectopic flowers. In contrast to FveLFYa, FveLFYb/c/d appear dispensable under normal growth conditions, as fvelfyc mutants are indistinguishable from wild type and FveLFYb and FveLFYd are barely expressed. Transgenic analysis and yeast one-hybrid assay showed that FveLFYa and FveLFYb, but not FveLFYc and FveLFYd, are functionally conserved with AtLFY in Arabidopsis (Arabidopsis thaliana). Unexpectedly, LFY-binding site prediction and yeast one-hybrid assay revealed that the transcriptional links between LFY and the APETALA1 (AP1) promoter/the large AGAMOUS (AG) intron are missing in F. vesca, which is due to the loss of LFY-binding sites. The data indicate that mutations in cis-regulatory elements could contribute to LFY evolution. Moreover, we showed that FveLFYa is involved in leaf development, as approximately 30% of mature leaves have smaller or fewer leaflets in fvelfya. Phylogenetic analysis indicated that LFY homologs in Fragaria species may arise from recent duplication events in their common ancestor and are undergoing convergent gene loss. Together, these results provide insight into the role of LFY in flower and leaf development in strawberry and have important implications for the evolution of LFY.

SWI2/SNF2 chromatin remodeling ATPases SPLAYED and BRAHMA control embryo development in rice.

Chromatin remodeling ATPases OsSYD and OsBRM are involved in shoot establishment, and both affect OSH gene transcription. OsSYD protein interacts with RFL, but OsBRM does not. In plants, SPLAYED (SYD) and BRAHMA (BRM) encode chromatin remodeling ATPases that use the energy derived from ATP hydrolysis to restructure nucleosomes and render certain genomic regions available to transcription factors. However, the function of SYD and BRM on rice growth and development is unknown. Here, we constructed ossyd and osbrm mutants using CRISPR/Cas9 technology and analyzed the effects of mutations on rice embryo development. We discovered that the ossyd and osbrm mutants exhibited severe defects during embryonic development, whereas endosperm development was normal. These results indicated that the development of the embryo and endosperm is independent of each other. Consequently, the ossyd- and osbrm-null mutants did not germinate due to the abnormal embryos. Furthermore, we observed the embryos of ossyd- and osbrm-null mutants, and they indeed had distinct differentiation defects in shoot establishment, acquired during embryogenesis. To verify the function of OsSYD and OsBRM in embryogenesis, we measured the transcript levels of marker genes at different stages. Compared with wild type, the expression levels of multiple OSH genes were significantly reduced in the mutants, which was consistent with the defective shoot establishment phenotypes. The interaction between SYD and RICE FLORICAULA/LFY (RFL) was revealed using a yeast two-hybrid screening system, suggesting that the interaction between the LFY homolog and chromatin remodeling ATPases is ubiquitous in plants. Collectively, our findings provide the basis for elucidating the function of OsSYD and OsBRM during embryo development in rice.

Evolution and expression of LEAFY genes in ferns and lycophytes

BackgroundThe LEAFY (LFY) transcription factors are present in algae and across land plants. The available expression and functional data of these genes in embryophytes suggest that LFY genes control a plethora of processes including the first zygotic cell division in bryophytes, shoot cell divisions of the gametophyte and sporophyte in ferns, cone differentiation in gymnosperms and floral meristem identity in flowering plants. However, their putative plesiomorphic role in plant reproductive transition in vascular plants remains untested.ResultsWe perform Maximum Likelihood (ML) phylogenetic analyses for the LFY gene lineage in embryophytes with expanded sampling in lycophytes and ferns. We recover the previously identified seed plant duplication that results in LEAFY and NEEDLY paralogs. In addition, we recover multiple species-specific duplications in ferns and lycophytes and large-scale duplications possibly correlated with the occurrence of whole genome duplication (WGD) events in Equisetales and Salviniales. To test putative roles in diverse ferns and lycophytes we perform LFY expression analyses in Adiantum raddianum, Equisetum giganteum and Selaginella moellendorffii. Our results show that LFY genes are active in vegetative and reproductive tissues, with higher expression in early fertile developmental stages and during sporangia differentiation.ConclusionsOur data point to previously unrecognized roles of LFY genes in sporangia differentiation in lycophytes and ferns and suggests that functions linked to reproductive structure development are not exclusive to seed plant LFY homologs.

Isolation and characterization of LEAFY homologous gene partial sequences from Dendrobium capra J.J. Smith Endemic Orchid of Java Island

Dendrobium capra is an endemic orchid species on the island of Java, Indonesia, which blooms only once a year, usually in February. In the flowering process, LEAFY (LFY) gene and its homologs are considered as key regulators in flowering of higher plants, including orchids. To understand floral regulatory mechanisms in orchids, information on the flowering gene structure needs to be explored. Partial sequences of LFY homologue of D. capra has been isolated and consists of 220 bp encoding 72 amino acid sequences. Bioinformatic analysis using Blastp search showed high similarity between the LFY genes of D. capra with other orchid LFY genes and has adjacent evolutionary relationship among orchid’sLFY. Amino acid sequence alignment result showed similar characteristic with other orchid’s LFY with conserved motif leucine-rich repeat and DDPRRRLLLLSPDQHN amino acid motif.

CsLFY is required for shoot meristem maintenance via interaction with WUSCHEL in cucumber (Cucumis sativus).

Cucumber (Cucumis sativus) is an agronomically important vegetable with indeterminant growth habit, in which leaves are produced from the shoot apical meristem (SAM), and unisexual flowers are generated from the leaf axils. LEAFY (LFY) and its homologs have been shown to play important roles in promoting flower development and branching. The LFY homolog gene CsLFY was cloned in cucumber. Molecular biology, developmental biology and biochemical tools were combined to explore the biological function of the LFY homologous gene CsLFY in cucumber. CsLFY was expressed in the SAM, floral meristem and floral organ primordia. Ectopic expression of CsLFY rescued the phenotype of the lfy-5 mutant in Arabidopsis. Knockdown of CsLFY by RNA interference (RNAi) led to defective shoot development and premature discontinuance of leaf initiation in cucumber. Transcription of CsWUS and putative CsLFY target genes including CsAP3 and CUM1 were significantly reduced in the CsLFY-RNAi lines. Further biochemical analyses indicated that CsLFY physically interacts with CsWUS in cucumber. These data suggested that CsLFY has a novel function in regulating shoot meristem maintenance through interaction with CsWUS, and promotes flower development via activation of CsAP3 and CUM1 in cucumber.

Two LEAFY homologs ILFY1 and ILFY2 control reproductive and vegetative developments in Isoetes L.

LEAFY (LFY) is a plant-specific transcription factor, which is found in algae and all land plants. LFY homologs exert ancestral roles in regulating cell division and obtain novel functions to control floral identity. Isoetes L. is an ancient genus of heterosporous lycophytes. However, characters about LFY homologs in lycophytes remain poorly investigated. In this study, two LFY homologs, ILFY1 and ILFY2, were cloned from five Isoetes species, including I. hypsophila, I. yunguiensis, I. sinensis, I. orientalis, and I. taiwanensis. The full length of ILFY1 was 1449–1456 bp with an open reading frame (ORF) of 927–936 bp. The full length of ILFY2 was 1768 bp with ORF of 726 bp. Phylogenetic tree revealed that ILFY1 and ILFY2 were separated into two clades, and I. hypsophila were separated with the others. Expression analysis demonstrated that IsLFY1 and IsLFY2 for I. sinensis did not show functional diversity. The two transcripts were similarly accumulated in both vegetative and reproductive tissues and highly expressed in juvenile tissues. In addition, the IsLFY1 and IsLFY2 transgenic Arabidopsis similarly did not promote precocious flowering, and they were inactive to rescue lfy mutants. The results facilitate general understandings about the characteristics of LFY in Isoetes and evolutionary process.

A link between LEAFY and B-gene homologues in Welwitschia mirabilis sheds light on ancestral mechanisms prefiguring floral development.

Flowering plants evolved from an unidentified gymnosperm ancestor. Comparison of the mechanisms controlling development in angiosperm flowers and gymnosperm cones may help to elucidate the mysterious origin of the flower. We combined gene expression studies with protein behaviour characterization in Welwitschia mirabilis to test whether the known regulatory links between LEAFY and its MADS-box gene targets, central to flower development, might also contribute to gymnosperm reproductive development. We found that WelLFY, one of two LEAFY-like genes in Welwitschia, could be an upstream regulator of the MADS-box genes APETALA3/PISTILLATA-like (B-genes). We demonstrated that, even though their DNA-binding domains are extremely similar, WelLFY and its paralogue WelNDLY exhibit distinct DNA-binding specificities, and that, unlike WelNDLY, WelLFY shares with its angiosperm orthologue the capacity to bind promoters of Welwitschia B-genes. Finally, we identified several cis-elements mediating these interactions in Welwitschia and obtained evidence that the link between LFY homologues and B-genes is also conserved in two other gymnosperms, Pinus and Picea. Although functional approaches to investigate cone development in gymnosperms are limited, our state-of-the-art biophysical techniques, coupled with expression studies, provide evidence that crucial links, central to the control of floral development, may already have existed before the appearance of flowers.

Dormancy release and flowering time in Ziziphus jujuba Mill., a “direct flowering” fruit tree, has a facultative requirement for chilling

In deciduous fruit trees, the effect of chilling on flowering has mostly been investigated in the “indirect flowering” group, characterized by a period of rest between flower bud formation and blooming. In the present study, we explored the effects of chilling and chilling deprivation on the flowering of Ziziphus jujuba, a temperate deciduous fruit tree belonging to the “direct flowering” group, in which flower bud differentiation, blooming and fruit development occur after dormancy release, during a single growing season. Dormancy release, vegetative growth and flowering time in Z. jujuba cv. Ben-Li were assessed following several treatments of chilling. Chilling treatments quantitatively decreased the timing of vegetative bud dormancy release, thereby accelerating flowering, but had no effect on the time from dormancy release to flowering. Trees grown at a constant temperature of 25°C, without chilling, broke dormancy and flowered, indicating the facultative character of chilling in this species. We measured the expression of Z. jujuba LFY and AP1 homologues (ZjLFY and ZjAP1). Chilling decreased ZjLFY expression in dormant vegetative buds but had no effect on ZjAP1expression, which reached peak expression before dormancy release and at anthesis.In conclusion, chilling is not obligatory for dormancy release of Z. jujuba cv. Ben-Li vegetative buds. However, the exposure to chilling during dormancy does accelerate vegetative bud dormancy release and flowering.

Cloning and characterization of the LFY homologue from Chinese cabbage (Brassica rapa subsp. pekinensis)

Flowering is critical to the growth and development of plants, and LFY gene homologues play a major role in flowering initiation. To understand the genetic and molecular mechanisms underlying floral initiation and development in Brassica rapa subsp. pekinensis, BrpLFY, a homologue of LFY, was cloned using RT-PCR. Sequence analysis showed that the cDNA sequence of BrpLFY is 1,341 bp in length, with an ORF of 1,245 bp encoding a predicted protein of 415 amino acids. The predicted protein showed a high degree of identity with LFY homologues from other angiosperm species. Real-time PCR analysis showed that BrpLFY mRNA was detected in all tissues during plant development from the vegetative state to fully differentiated flowers, and its expression was highest in the cotyledon and lowest in the root. BrpLFY expression in the shoot apex increased gradually during vegetative growth and increased dramatically at stage 1 of flower bud differentiation. The relative expression peaked at stage 5 and then decreased in later stages. Moreover, the trend in BrpLFY expression level change in the shoot apex was similar regardless of variety or vernalization method. The relative expression of BrpLFY in leaves gradually decreased with leaf development. We overexpressed the gene in Arabidopsis thaliana using the floral dip method, and examined flowering time in wild-type and transgenic plants. Overexpression of BrpLFY specifically caused early flowering; the transgenic plants flowered 10-14 d earlier than did wild-type plants, and leaf number decreased by 0.5-1 when the plants bolted. Real-time PCR analysis showed that the expression of BrpLFY in transgenic Arabidopsis was higher than in wild-type plants. These results indicate that BrpLFY plays a role in promoting flowering in Chinese cabbage.

Functional Characterization ofPhapLEAFY, aFLORICAULA/LEAFYOrtholog inPhalaenopsis aphrodite

The plant-specific transcription factor LEAFY (LFY) is considered to be a master regulator of flower development in the model plant, Arabidopsis. This protein plays a dual role in plant growth, integrating signals from the floral inductive pathways and acting as a floral meristem identity gene by activating genes for floral organ development. Although LFY occupies an important position in flower development, the functional divergence of LFY homologs has been demonstrated in several plants including monocots and gymnosperms. In particular, the functional roles of LFY genes from orchid species such as Phalaenopsis that contain unique floral morphologies with distinct expression patterns of floral organ identity genes remain elusive. Here, PhapLFY, an ortholog of Arabidopsis LFY from Phalaenopsis aphrodite subsp. formosana, a Taiwanese native monopodial orchid, was isolated and characterized through analyses of expression and protein activity. PhapLFY transcripts accumulated in the floral primordia of developing inflorescences, and the PhapLFY protein had transcriptional autoactivation activity forming as a homodimer. Furthermore, PhapLFY rescues the aberrant floral phenotypes of Arabidopsis lfy mutants. Overexpression of PhapLFY alone or together with PhapFT1, a P. aphrodite subsp. formosana homolog of Arabidopsis FLOWERING LOCUS T (FT) in rice, caused precocious heading. Consistently, a higher Chl content in the sepals and morphological changes in epidermal cells were observed in the floral organs of PhapLFY knock-down orchids generated by virus-induced gene silencing. Taken together, these results suggest that PhapLFY is functionally distinct from RICE FLORICAULA/LEAFY (RFL) but similar to Arabidopsis LFY based on phenotypes of our transgenic Arabidopsis and rice plants.

Genetic diversity of LEAFY homologues in Chrysanthemum argyrophyllum L

The LEAFY (LFY) gene encodes a key transcription factor involved in flower development. In this study, we identified the LFY homolog in Chrysanthemum argyrophyllum L. (CaFL) and sequenced eight clones from a single plant. The cloned genes varied in length (2915–3075 bp) and each had three exons and two introns. The three exons were highly conserved, so intron variance was primarily responsible for gene length differences. The putative CaFL proteins from the eight clones were 99% identical to one another and greater than 97% identical to Chrysanthemum lavandulifolium DFL and Chrysanthemum nankingense CnFL. Intraspecific and interspecific sequence comparisons of the two introns indicated that they varied considerably in both length and sequence composition. Phylogenetic analysis showed that CaFL is more similar to dicotyledonous FLO/LFY-like proteins than to those from monocotyledons or gymnosperms.

Characterization of the sequence and expression pattern of LFY homologues from dogwood species (Cornus) with divergent inflorescence architectures

LFY homologues encode transcription factors that regulate the transition from vegetative to reproductive growth in flowering plants and have been shown to control inflorescence patterning in model species. This study investigated the expression patterns of LFY homologues within the diverse inflorescence types (head-like, umbel-like and inflorescences with elongated internodes) in closely related lineages in the dogwood genus (Cornus s.l.). The study sought to determine whether LFY homologues in Cornus species are expressed during floral and inflorescence development and if the pattern of expression is consistent with a function in regulating floral development and inflorescence architectures in the genus. Total RNAs were extracted using the CTAB method and the first-strand cDNA was synthesized using the SuperScript III first-strand synthesis system kit (Invitrogen). Expression of CorLFY was investigated by RT-PCR and RNA in situ hybridization. Phylogenetic analyses were conducted using the maximum likelihood methods implemented in RAxML-HPC v7.2.8. cDNA clones of LFY homologues (designated CorLFY) were isolated from six Cornus species bearing different types of inflorescence. CorLFY cDNAs were predicted to encode proteins of approximately 375 amino acids. The detection of CorLFY expression patterns using in situ RNA hybridization demonstrated the expression of CorLFY within the inflorescence meristems, inflorescence branch meristems, floral meristems and developing floral organ primordia. PCR analyses for cDNA libraries derived from reverse transcription of total RNAs showed that CorLFY was also expressed during the late-stage development of flowers and inflorescences, as well as in bracts and developing leaves. Consistent differences in the CorLFY expression patterns were not detected among the distinct inflorescence types. The results suggest a role for CorLFY genes during floral and inflorescence development in dogwoods. However, the failure to detect expression differences between the inflorescence types in the Cornus species analysed suggests that the evolutionary shift between major inflorescence types in the genus is not controlled by dramatic alterations in the levels of CorLFY gene transcript accumulation. However, due to spatial, temporal and quantitative limitations of the expression data, it cannot be ruled out that subtle differences in the level or location of CorLFY transcripts may underlie the different inflorescence architectures that are observed across these species. Alternatively, differences in CorLFY protein function or the expression or function of other regulators (e.g. TFL1 and UFO homologues) may support the divergent developmental trajectories.

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. • Cloned a LFY / FLO homolog gene CnFL from an absolute plant Chrysanthemum nankingense . • Construction the phylogenetic tree using the predicted protein among LFY homologs. • The expression of CnFL during the photoperiodic flower induction indicated that the CnFL was up-regulated by photoperiodic.

COCHLEATA controls leaf size and secondary inflorescence architecture via negative regulation of UNIFOLIATA (LEAFY ortholog) gene in garden pea Pisum sativum.

UNIFOLIATA [(UNI) or UNIFOLIATA-TENDRILLED ACACIA (UNI-TAC)] expression is known to be negatively regulated by COCHLEATA (COCH) in the differentiating stipules and flowers of Pisum sativum. In this study, additional roles of UNI and COCH in P. sativum were investigated. Comparative phenotyping revealed pleiotropic differences between COCH (UNI-TAC and uni-tac) and coch (UNI-TAC and uni-tac) genotypes of common genetic background. Secondary inflorescences were bracteole-less and bracteolated in COCH and coch genotypes, respectively. In comparison to the leaves and corresponding sub-organs and tissues produced on COCH plants, coch plants produced leaves of 1.5-fold higher biomass, 1.5-fold broader petioles and leaflets that were 1.8-fold larger in span and 1.2-fold dorso-ventrally thicker. coch leaflets possessed epidermal cells 1.3-fold larger in number and size, 1.4-fold larger spongy parenchyma cells and primary vascular bundles with 1.2-fold larger diameter. The transcript levels of UNI were at least 2-fold higher in coch leaves and secondary inflorescences than the corresponding COCH organs. It was concluded that COCH negatively regulated UNI in the differentiating leaves and secondary inflorescences and thereby controlled their sizes and/or structures. It was also surmised that COCH and UNI (LFY homolog) occur together widely in stipulate flowering plants.

Cloning and expression analysis of a LFY homologous gene in Chinese jujube (Ziziphus jujube Mill.)

LEAFY (LFY) homologous genes are necessary for the transition from vegetative to reproductive development in flowering plants. The full-length cDNA of a LFY homolog was successfully isolated from floral buds of Chinese Jujube ( Ziziphus jujube Mill.) by degenerate reverse transcriptase-polymerase chain reaction (RT-PCR), rapid amplification of cDNA ends (RACE), and Blastn analysis. The LFY homologous gene was denominated as ZjLFY and deposited at the GenBank under accession JN165097. The full-length cDNA sequence of ZjLFY was 1517 bp in length and contained a completed open reading frame (ORF) of 1209 bp encoding 576 amino acids. Amino acid sequence comparison revealed that the ZjLFY had two variable regions and two conserved regions. At the amino acid level, the ZjLFY showed high similarity ranging from 57.5 to 82.9% with other LFY homologous members. Semi-quantitative RT-PCR and quantitative real-time PCR analysis revealed strong expression of ZjLFY in floral buds, but weak expression in fruit-bearing shoots, vegetative shoots, stems, mature leaves and axillary buds, suggesting that the ZjLFY played a role in both vegetative and reproductive development in Chinese jujube.

Seasonal changes in the expression pattern of flowering-related genes in evergreen azalea ‘Oomurasaki’ ( Rhododendron × pulchrum)

To investigate seasonal changes in transcript levels of flowering-related genes corresponding to shoot condition, the endogenous expression patterns of FLOWERING LOCUS T ( FT), SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 ( SOC1), LEAFY ( LFY), TERMINAL FLOWER 1 ( TFL1), and CENTRORADIALIS ( CEN) homologs were investigated in evergreen azalea ‘Oomurasaki’. In this plant, in which the floral transition is triggered by the about 25 °C and shortening days of summer under natural conditions, the mRNA levels of the FT, SOC1, and LFY homologs ( RpFT, RpSOC1, and RpLFY) increased in August corresponding to the period of flower bud formation, and the mRNA levels of TFL1 and CEN homologs ( RpTFL1 and RpCEN) increased during July before flower bud formation. RpTFL1 showed low transcript levels during the period of flower bud formation and development, but RpCEN showed a different expression pattern from RpTFL1. The expression of RpCEN, RpSOC1, and RpLFY peaked again during January corresponding to the period of flower bud dormancy. During blooming in May, the expression levels of all the investigated genes were low. In the period of flower bud formation, RpFT expression started to increase, and the expression of RpSOC1 peaked in mid-July. These results suggest that the shoot apical meristem (SAM) starts to transition from vegetative to reproductive growth in mid-July. Thus, changes in the transcription of RpFT, RpSOC1, and RpLFY were found to correlate positively with seasonal flowering in the shoot apex of ‘Oomurasaki’, whereas changes in RpTFL1 and RpCEN transcription correlated negatively with seasonal flowering.

Molecular cloning and sequence analysis of an LFY homologous gene from Juglans regia L.

The existence of a long juvenile phase is found seriously to affect the early-stage economic benefits of later mature walnuts (Juglans regia L.). Studies on LFY, a key gene controlling floral transition and flower differentiation, may be helpful in solving the problem. This study reports the identification and characterization of a JrLFY gene from Juglans regia L., a homolog of FLORICAULA/LFY. The gene was isolated from flower buds of precocious walnut cultivar Zhonglin No. 5 by RT-PCR and RACE. The cDNA sequence of JrLFY (GenBank accession no. GU194836) was 1496 bp and contained an ORF of 1158 bp. Its corresponding genomic sequence (GenBank accession no. HQ019159) showed that the JrLFY contained three exons and two introns. The predicted amino acid sequence of the gene consisted of 385 amino acids and had a conserved region in the C-terminal when being aligned with sequences of other LFY homologs. Phylogenetic analysis showed that the LFY protein of walnut was close to those of hickory and chestnut. These studies will lay a foundation for understanding the mechanism of early fruiting and preparation for transfer of the JrLFY as a transgene to later mature walnuts.

Prediction of Regulatory Interactions from Genome Sequences Using a Biophysical Model for theArabidopsisLEAFY Transcription Factor

Despite great advances in sequencing technologies, generating functional information for nonmodel organisms remains a challenge. One solution lies in an improved ability to predict genetic circuits based on primary DNA sequence in combination with detailed knowledge of regulatory proteins that have been characterized in model species. Here, we focus on the LEAFY (LFY) transcription factor, a conserved master regulator of floral development. Starting with biochemical and structural information, we built a biophysical model describing LFY DNA binding specificity in vitro that accurately predicts in vivo LFY binding sites in the Arabidopsis thaliana genome. Applying the model to other plant species, we could follow the evolution of the regulatory relationship between LFY and the AGAMOUS (AG) subfamily of MADS box genes and show that this link predates the divergence between monocots and eudicots. Remarkably, our model succeeds in detecting the connection between LFY and AG homologs despite extensive variation in binding sites. This demonstrates that the cis-element fluidity recently observed in animals also exists in plants, but the challenges it poses can be overcome with predictions grounded in a biophysical model. Therefore, our work opens new avenues to deduce the structure of regulatory networks from mere inspection of genomic sequences.

Expression of a FLOWERING LOCUS T homologue is temporally associated with annual flower bud initiation in Eucalyptus globulus subsp. globulus (Myrtaceae)

The transition to flowering in plants is the result of the balance of endogenous processes and environmental signals that act through a complex genetic pathway that has been studied extensively in annual plants such as Arabidopsis. Perennial trees are characterised by a juvenile non-flowering phase lasting several years followed by an adult phase in which there is repeated cycling between vegetative and reproductive growth. The genetic control of flowering time is potentially more complex in perennials than in annuals and is less understood. Here, we examine the control of flowering in Eucalyptus globulus subsp. globulus, an important forestry species in temperate parts of the world. The E. globulus subsp. globulus homologues of two important flowering genes FLOWERING LOCUS T (FT) and LEAFY (LFY) were isolated and quantitative RT-PCR was used to measure their expression over a 2-year period. The expression of the homologue of FT in E. globulus subsp. globulus leaves was associated with the annual transition from vegetative to reproductive growth (i.e. flower bud initiation). Expression of the LFY homologue was associated with early flower bud development. In a comparison of FT and LFY expression patterns in two clones each of an early and late anthesis genotype, no association between the expression of these genes and the timing of anthesis was shown. Taken together, this indicates that FT and LFY could form part of the flower initiation pathway in Eucalyptus but do not regulate the observed differences in anthesis time.

Isolation of a LEAFY homolog from Populus tomentosa: expression of PtLFY in P. tomentosa floral buds and PtLFY-IR-mediated gene silencing in tobacco (Nicotiana tabacum)

To understand the genetic and molecular mechanisms underlying floral development in Populus tomentosa, we isolated PtLFY, a LEAFY homolog, from a P. tomentosa floral bud cDNA library. DNA gel blot analysis showed that PtLFY is present as a single copy in the genomes of both male and female individuals of P. tomentosa. The genomic copy is composed of three exons and two introns. Relative expression levels of PtLFY in tissues of P. tomentosa were estimated by RT-PCR; our results revealed that PtLFY mRNA is highly abundant in roots and both male and female floral buds. A low level of gene expression was detected in stems and vegetative buds, and no PtLFY-specific transcripts were detected in leaves. PtLFY expression patterns were analyzed during the development of both male and female floral buds in P. tomentosa via real-time quantitative RT-PCR. Continuous, stable and high-level expression of PtLFY-specific mRNA was detected in both male and female floral buds from September 13th to February 25th, but the level of PtLFY transcripts detected in male floral buds was considerably higher than in female floral buds. Our results also showed an inverted repeat PtLFY fragment (PtLFY-IR) effectively blocked flowering of transgenic tobacco plants, and that this effect appeared to be due to post-transcriptional silencing of the endogenous tobacco LFY homologs NFL1 and NFL2.