JXB Advance Access originally published online on May 23, 2005
Journal of Experimental Botany 2005 56(417):1965-1974; doi:10.1093/jxb/eri194
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Published by Oxford University Press [2005] on behalf of the Society for Experimental Biology.
RESEARCH PAPER |
The rubber tree (Hevea brasiliensis Muell. Arg.) homologue of the LEAFY/FLORICAULA gene is preferentially expressed in both male and female floral meristems*
Universidade de São Paulo, Centro de Energia Nuclear na Agricultura, Laboratório de Biotecnologia Vegetal, Av. Centenário, 303 CEP 13400-970 Piracicaba, SP, Brazil
Present address and to whom correspondence should be sent: Universidade Estadual de Campinas, Instituto de Biologia, Departamento de Fisiologia Vegetal, Cidade Universitária "Zeferino Vaz", 13084-971 Campinas, SP, Brazil. E-mail: mcdornel{at}cena.usp.br
Received 9 December 2004; Accepted 7 April 2005
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Abstract |
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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.
Key words: Development, flowering, gene expression, LEAFY, plant reproduction, rubber tree
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The knowledge of floral ontogeny in plant species is essential for the establishment of breeding programmes and for the understanding of the evolutionary processes involved in the development of the floral organs. Regulation of flowering in woody perennials shows remarkable differences with respect to herbaceous species, i.e. long juvenile phases, season-dependent bud dormancy, and the need for alternating vegetative and reproductive phases. Despite the interest in these processes for the management and improvement of woody species, very little is known about their underlying molecular mechanisms. Genetic and molecular approaches in herbaceous species such as snapdragon (Antirrhinum majus) and Arabidopsis have allowed the identification of some of the key genes regulating flowering induction and reproductive development (Pidkowich et al., 1999
; Simpson et al., 1999; Araki, 2001). Among them, FLORICAULA (FLO) from snapdragon (Carpenter and Coen, 1990; Coen et al., 1990) and its Arabidopsis orthologue, LEAFY (LFY); Schultz and Haughn, 1991; Weigel et al., 1992), seem to have a central role in the specification of flower meristem identity. Inactivation of FLO causes the development of indeterminate shoots in place of flowers (Coen et al., 1990), whereas lfy mutants show partial flower-to-shoot conversions (Weigel et al., 1992).
Constitutive expression of LFY is sufficient to promote flower initiation and development from shoot apical and axillary meristems in Arabidopsis and has similar effects in other dicot and monocot species (Weigel and Nilsson, 1995; He et al., 2001; Peña et al., 2001), suggesting a conservation of LFY function among distantly related species within angiosperms. Despite FLO/LFY sequence conservation (Frohlich and Parker, 2000), significant differences are emerging in relation to their expression patterns that could indicate the existence of a functional divergence. For instance, although, in snapdragon, expression of FLO is specific of the reproductive phase (bracts and young floral meristems; Coen et al., 1990), low levels of expression of FLO/LFY orthologues have been detected in leaf primordia during vegetative growth in Arabidopsis, tobacco (Nicotiana tabacum), Impatiens sp., pea (Pisum sativum), petunia (Petunia hybrida), and tomato (Lycopersicon esculentum; Kelly et al., 1995; Blázquez et al., 1997; Bradley et al., 1997; Hofer et al., 1997; Pouteau et al., 1997; Souer et al., 1998; Molinero-Rosales et al., 1999). Consistent with this evidence, a role in leaf development has been proposed for pea and tomato FLO/LFY orthologues based on the morphological alterations shown in the leaves of loss-of-function mutants. The expression patterns of monocot FLO/LFY orthologues are even more divergent from those observed for their dicots counterparts. In rice (Oryza sativa), RLF gene expression is restricted to young panicles (Kyozuka et al., 1998). In Arabidopsis and Antirrhinum FLO/LFY expression precedes SQUA/AP1 activation and LFY is required for AP1 up-regulation (Liljegren et al., 1999; Wagner et al., 1999). By contrast, in Lolium temulentum, LtLFY is expressed later than the SQUA/AP1 orthologue, LtMADS2 (Gocal et al., 2001).
Some FLO/LFY orthologues have been cloned and characterized in woody species such as eucalyptus (Eucalyptus globulus; Southerton et al., 1998), Monterey pine (Pinus radiata; Mellerowicz et al., 1998; Mouradov et al., 1998), Populus trichocarpa (Rottmann et al., 2000), kiwifruit (Actinidia deliciosa; Walton et al., 2001), and apple (Wada et al., 2002). However, the specific roles of these genes in controlling the characteristic features of tree reproductive development are still being elucidated. Furthermore, partial or total FLO/LFY-like sequences have been reported from other basal angiosperms and gymnosperms (Frohlich and Meyerowitz, 1997; Frohlich and Parker, 2000) although, in these cases, functional information is not available.
The authors were interested in the reproductive development of rubber tree (Hevea brasiliensis Muell. Arg.), an important source of natural rubber that belongs to the Euphorbiaceae family (Judd et al., 1999). Rubber trees grown from seeds go through 5–8 years of a juvenile phase before they start to flower (Cuco and Bandel, 1998). The rubber tree, similar to other Euphorbiaceae species, produces lateral inflorescences containing male and female flowers (Webster, 1994) and, recently, hermaphroditism was described to occur in some commercial varieties, such as the economically important commercial variety RRIM600 (Cuco and Bandel, 1998). To understand the genetic and molecular mechanisms underlying the flowering process in the rubber tree, the development of buds was analysed by scanning electron microscopy (SEM) during two growing seasons and their development has been related to the temporal and spatial expression patterns of HbLFY, the rubber tree FLO/LFY orthologue gene. Cloning and characterization of HbLFY indicate that it is a single-copy gene, as in other angiosperm species. In situ hybridization experiments shows that HbLFY is expressed in lateral meristems depending on their meristematic fate. The HbLFY transcripts were observed only in reproductive buds and in the early stages of male, female, and hermaphrodite flower meristem development. These results could suggest that HbLFY is involved in inflorescence development as well as in flower initiation.
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Materials and methods |
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Plant material
Samples of vegetative and reproductive tissues of Hevea brasiliensis RRIM600 were collected in the fields of the Escola Superior de Agricultura ‘Luiz de Queiroz’, at the University of São Paulo (Piracicaba, SP, Brazil) during the years 2000 to 2003. Young expanding leaves were also used for the extraction of genomic DNA. Scanning electron microscopy (SEM), total RNA extraction, and in situ hybridization were performed on the plant tissues collected and fixed in different developmental stages. For the in situ hybridization experiments, meristems from both adult (more than 16-year-old) and juvenile (up to 2-months-old) plants were collected.
Library construction and cloning of HbLFY
Genomic DNA for PCR amplification, Southern analysis, and construction of genomic libraries was isolated by the traditional CTAB-based method (Sambrook et al., 1989). Total RNA for cDNA library construction and northern blot was isolated from rubber tree leaves, vegetative apices, and from a mix of inflorescences at different developmental stages using the Rneasy kit (Qiagen) following the supplier's instructions.
The genomic clones of HbLFY were isolated by screening 600 000 plaques from a H. brasiliensis genomic library (61x106 pfu, using the Packagene Lambda Packing Systems from Promega, USA) constructed with partially Sau3A-digested genomic DNA. For this screening, a biotin-labelled probe was used (North2South chemiluminescent system, Pierce, USA) using the entire Arabidopsis LFY cDNA from plasmid pDW124 (Weigel et al., 1992) as a template. Three adjacent EcoRI-BamHI, BamHI-EcoRI, and EcoRI-EcoRI fragments (H5EB, H3BE, and H4E, with 6.1, 2.1, and 3.25 kbp, respectively) containing the genomic HbLFY sequence were subcloned into pBluescriptKS (Clontech). Subclones were prepared by nested deletions (Zhu and Clark, 1995) and sequenced on an ABI Prism 377 (Perkin-Elmer/Applied Biosystems) automated sequencer using the DYEnamic ET terminator Cycle Sequencing Kit (Amersham/Pharmacia Biotec, USA) coupled with M13 reverse and forward primers following the manufacturer's instructions.
Total RNA from a mix of inflorescences at different developmental stages was used to construct a cDNA library. The poly-A fraction of RNA was isolated and the first strand of cDNA was synthesized using the SuperScript cloning system (Life Technologies). The cDNA library screening was performed by a PCR-based strategy (Sussman et al., 2000) using the LFY-specific degenerate primers L1: 5'-CGGAYATIAAYAARCCIAARATGMGICAYTA-3' and L4: 5'-CGGATCCGTGICKIARIYKIGTIG-GIACRTA-3' (Frohlich and Meyerowitz, 1997). The insert size of the positive clones were determined by PCR using the M13 forward and reverse primers and the four longest clones were sequenced on both strands.
The partial HbLFY sequences obtained from cDNA and genomic subclones were trimmed from vector sequences and assembled, before being checked for similarity with sequences already deposited in public databases using BLASTX (Altschul et al., 1997).
The genomic and cDNA sequences of the HbLFY gene were deposited in GenBank under the accession numbers AY639378 and AY639379, respectively.
DNA and RNA blot hybridization
Genomic Southern blotting was performed as described in Sambrook et al. (1989) using genomic DNA digested with XhoI and PstI and blotted on the Hybond-N+ membrane (Amersham). Northern experiments were performed using 10 µg of total RNA extracted from leaves, vegetative apices, and from a mix of inflorescences at different developmental stages, separated in a denaturing agarose gel (Sambrook et al., 1989), and hybridized to a fluorescein-labelled HbLFY probe.
The HbLFY probe used in both Southern and northern experiments was a 235 bp PCR product obtained from the 3' transcribed region of the gene, using primers E13: 5'-TGGCGGAGCTTGGTGGGGACA-3' and E25: 5'- CTTCCTCCTCCAAGTCCAATC-3', using a HbLFY cDNA clone as template. PCR reactions were performed in a final volume of 25 µl with an initial 3 min denaturation at 96 °C, followed by 40 cycles of 96 °C for 40 s; 45 °C for 30 s, and 72 °C for 2 min. The PCR product was purified using the Concert Kit (Gibco-Life Sciences). The probe was labelled with fluorescein using the DCP-Star GeneImage System (Pharmacia-Amersham). Hybridization conditions, washing stringencies and detection were those suggested by the kit manufacturer. As a control for gel loading in northern experiments, the stripped membrane was rehybridized with a heterologous probe for a constitutively expressed gene, under low stringency, using a cDNA for an Arabidopsis ubiquitin (GenBank accession AB5432) as template. Previoulsy, this heterologous probe was tested in a Southern blot (prepared with H. brasiliensis total DNA digested with XhoI or PstI) under low stringency and shown to hybridize to a single band (data not shown).
Characterization of HbLFY expression patterns by in situ hybridization
Slide preparation, digoxigenin labelling of RNA probes, and hybridization conditions were performed as described by Dornelas et al. (1999, 2000). The template for the HbLFY digoxigenin-labelled riboprobes was a 1.134 bp fragment, containing the complete HbLFY coding region, cloned in the pGEM-T easy vector. The hybridized sections were observed and photographed under a Zeiss Axiovert 35 microscope.
Scanning electron microscopy (SEM)
H. brasiliensis inflorescences at different developmental stages, as well as vegetative meristems, were immediately fixed in 4% paraformaldehyde under vacuum for 24 h, dehydrated in absolute ethanol, and stored at 4 °C until needed. For SEM observation, the plant material was initially dissected in absolute ethanol under an Olympus dissecting microscope. The resultant material was critical point-dried with CO2 in a Balzer's drier and further dissected, when necessary. The samples were mounted in metallic stubs with carbon conductive adhesive tape, coated with colloidal gold (40 nm thick) and observed at 10–20 kV using a ZEISS DSM 940 A or a LEO 435 VP scanning electron microscope, at the University of São Paulo (ESALQ-NAP/MEPA).
Phylogenetic analyses
Nucleotide and protein sequences of different LFY homologues were retrieved from GenBank (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi) and aligned with Clustal W (Thompson et al., 1994). Genetic distance matrixes were obtained from the alignments using MEGA (http://www.megasoftware.net) and Neigbor–Joining trees were built, with bootstrap sampling of 1000. Tree topologies were optimized using TreeView (Page, 2000).
Assessment of HbLFY biological activity using transgenic Arabidopsis
The XbaI–SmaI HbLFY fragment, carrying the coding region of HbLFY, with its endogenous start and stop codons, was obtained from plasmid pHBLFY and blunt-ended using DNA polymerase I (Klenow fragment). An intermediate pDW132H vector was prepared by cloning the polished fragment described above into the SmaI site of pDW132, containing the Arabidopsis LFY promoter (Weigel et al., 1992). The correct orientation of the cloning process was checked by endonuclease digestion. The PstI-SpeI fragment from the resultant pDW132H (LFY::HbLFY) vector was blunt-ended with Klenow and cloned into the plant transformation vector pSKI015 (a gift from D Weigel, Salk Insitute, LA Jolla CA, USA), which contains the bar gene, allowing selection with the herbicide Basta (Sylvet), constituting the pSKI015H vector. Arabidopsis plants (Columbia ecotype) transgenic for pSKI015H T-DNA were generated by using Agrobacterium tumefaciens-mediated in planta transformation as described by Bechtold and Pelletier (1998). Putatively transformed seeds were selected upon germination on sand wetted with a Basta (Sylvet) solution at 500 µl l–1. Homozygous (Basta-resistant) lines were created by selfing. The segregation ratio of resistant:sensitive was used to estimate the number of transformed T-DNA loci. T2 lines, homozygous for the LFY::HbLFY T-DNA loci, were identified by sowing 200–300 T2 seeds, derived from different T1 plants under selective conditions. Transgenic and non-transgenic plants were grown in growth chambers at 23 °C under illumination with fluorescent lights: long-day (LD) conditions (16/8 h light/dark) or short-day (SD) conditions (8/16 h light/dark). Finally, LFY::HbLFY transformants in the Columbia ecotype were crossed to the strong lfy-26 mutant allele in the Landsberg erecta background (wild-type and mutant Arabidopsis seeds were obtained from the ABRC seed stock at the Ohio State University facility at Columbus, Ohio, USA). To genotype F2 plants at the LFY locus, CAPS (Cleared Amplified Polymorphic Sequences; Konieczny and Ausubel, 1993) markers that distinguished between Columbia and Landsberg were used (URL:http://www.salk.edu/LABS/pbio-w/caps.html). Transgenic and non-transgenic Arabidopsis flowers and inflorescences at different developmental stages were photographed under a stereomicroscope or were analysed by SEM.
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Results |
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Reproductive development in H. brasiliensis
The H. brasiliensis inflorescences are determinate panicles formed upon the conversion of terminal vegetative meristems into inflorescence meristems. In the RRIM600 clone, the inflorescence may be terminated by a hermaphrodite (bisexual) flower (Cuco and Bandel, 1998
), although hermaphrodite flowers have been observed only sporadically in this study's material. The first open flowers are generally observed by September–October (mid-spring). Nevertheless, the first inflorescence meristems are already differentiated by late July to early August (Fig. 1A, B). The floral meristems are formed acropetally and are initiated on the periphery of the inflorescence meristem, being protected by bracts (Fig. 1C, D).
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The floral meristems (Fig. 1E) are organized in the main and secondary inflorescence axes. The floral organ differentiation occurs centripetally and is initiated with the differentiation of the abaxial tepal primordium (sepals and petals can not be differentiated), followed by the other tepal primordia (Fig. 1E). Five tepal promordia are formed in the outer whorl. In the RRIM600 clone, most of the flowers formed along the inflorescence axis are male and the female flowers occupy the terminal positions, except the terminal flower on the main axis where hermaphrodite flowers have also been reported to occur (Cuco and Bandel, 1998
). In the centre of the male flower meristem, ten stamen primordia are differentiated into two concentric rings over a columnar structure, the androphore (Fig. 1F, G). In the female flowers, three carpel primordia are formed, originally free, but, as they elongate, they undergo post-genital fusion, forming the ovary and the stigma as the flower develops (Fig. 1H, I). At the anthesis, a nectary is present at the base of the ovary and trichomes can be observed on the ovary epidermis (Fig. 1H, I).
Cloning and sequence analyses of HbLFY
The coding region of HbLFY is 1134 bp encoding 377 amino acids. The longest HbLFY cDNA clone showed a 77 bp 5' non-coding region and a 125 bp 3' non-coding region. The comparison of the cDNA and genomic sequences allowed the gene structure to be proposed (Fig. 2). HbLFY has two introns of 466 and 770 pb, respectively. The positions of the HbLFY introns are conserved when compared with other LFY homologues (Frohlich and Parker, 2000). A comparison of the amino acid sequences of HbLFY and other FLO/LFY homologues (Arabidopsis LFY; Weigel et al., 1992; snapdragon FLO, Coen et al., 1990; poplar, PTLF; Rottmann et al., 2000; pea, PEAFLO; Hofer et al., 1997; petunia, ALF; Souer et al., 1998; and tomato, TOFL; Molinero-Rosales et al., 1999) showed the presence of several conserved regions. The LFY, PFL, ALF, and TOFL proteins have each been described to have a proline-rich region (roughly the first 40 amino acids), but HbLFY showed a mixed proline- and alanine-rich region instead (data not shown). The phylogenetic tree of the FLO/LFY homologues (Fig. 3) clearly shows that duplicated homologues in angiosperms are relatively recent events (e.g. as in tobacco and maize), with the exception of a duplication that predated species divergence within Maloideae.
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Genomic DNA extracted from rubber trees was digested with rarely cutting enzymes (XhoI, and PstI), and probed at high stringency with a fluorescein-labelled probe. Figure 4A shows that there is only one band in both digestions, indicating that HbLFY is a single gene in the H. brasiliensis genome.
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Expression patterns of HbLFY
To detect the rough expression pattern of HbLFY during rubber tree development, a northern blot analysis was performed. Ten micrograms of total RNA was blotted to a nylon membrane and hybridized with a labelled HbLFY-specific probe (Fig. 4B). Figure 4B shows that the expression of HbLFY is restricted to developing inflorescences. To investigate the HbLFY expression patterns further, sense and anti-sense HbLFY probes were hybridized to longitudinal sections of vegetative and reproductive meristems at different developmental stages (Fig. 5). It was observed that the HbLFY mRNA can not be detected in vegetative meristems of juvenile (2-months-old) and adult (more than 16-years-old) plants (Fig. 5A–C). The accumulation of HbLFY transcripts was restricted to reproductive meristems and the developing floral organs (Fig. 5D–G). The HbLFY expression patterns agree with a putative role of HbLFY in rubber tree reproductive development.
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The HbLFY coding region can complement transgenic Arabidopsis-lfy mutants
The HbLFY coding region fused downstream to the Arabidopsis LFY promoter was used to obtain transgenic homozygous lfy-26 Arabidopsis mutant lines. Complete restoration of the wild-type phenotype was observed in all ten transgenic lines analysed (Fig. 6). The identity of the homozygous transgenic mutant plants was verified by CAPs genotyping (data not shown) that allows the distinction between the different ecotype backgrounds (Konieczny and Ausubel, 1993
). In the Arabidopsis lfy-26 mutants, the early arising (basal) flowers are replaced by bracts with secondary inflorescence shoots, whereas flowers arising later were replaced by small bracts, in whose axils abnormal flowers developed (compare Fig. 6A–D and see Weigel et al., 1992). These abnormal flowers contained sepals and carpels, but no petals or stamens, these later being usually homeotically substituted by more sepals and carpels, respectively (Fig. 6D; Weigel et al., 1992). By contrast, wild-type flowers typically contain four sepals, four petals, six stamens, and two carpels. The lfy-26 floral phenotype was largely complemented by the LFY::HbLFY transgene (Fig. 6E). The main inflorescence axis of these plants developed 2–3 secondary inflorescences in basal positions and solitary flowers at apical positions, and most of the flowers contained all four floral organ types.
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Discussion |
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The Euphorbiaceae family is extremely diverse and considered to be polyphyletic (Webster, 1994
). Thus the reproductive development habits within the family are also variable. Despite the fact that H. brasiliensis is the main source of most natural rubber, breeding programmes are sparse and mainly centred in clonal selection due to the long juvenile phase and the long generation time. It would be of great interest to regulate the flowering process in this woody perennial artificially, aiming at a more efficient breeding programme and conservation strategies. Regulation of flowering in woody perennials shows remarkable differences with respect to herbaceous species, i.e. long juvenile phases, bud dormancy, and alternating vegetative and reproductive development.
H. brasiliensis is a monoecious species with male, female, and bisexual flowers and flowering occurs annually or bi-annually in 5–8-year-old trees (Cuco and Bandel, 1998). The descriptions of floral development in species belonging to the Euphorbiaceae family are mostly limited to the species where the typical cyathium-type of inflorescence occurs (Judd et al., 1999; Webster, 1994). The main events of floral development in H. brasiliensis RRIM600 are described here. Some characteristics such as the formation of pentamerous flowers and a tricarpelar ovary are typical of the Euphorbiaceae family. The alternation in vegetative and reproductive growth in rubber trees by the formation of lateral inflorescences at the end of the dry season, implies a tight control of flowering time.
Distinct sets of genes that integrate environmental cues and activate the underlying molecular regulation of flower development have been characterized in model herbaceous plants, such as Arabidopsis. Among the earliest acting genes are the floral meristem identity genes, such as LFY, that are thought to establish and maintain the floral state, as opposed to the vegetative shoot fate, in apical and lateral meristems. Assuming that the basic mechanisms for floral regulation would be, at least partially, conserved among dicots, an expressed LFY homologue has been isolated from the rubber tree, HbLFY.
Southern blot results using endonuclease-digested rubber tree genomic DNA (Fig. 4A) indicated that its genome has only one gene homologous to LFY. While gymnosperm species have two homologues of FLO/LFY genes, angiosperm species have been shown to possess generally only one (Frohlich and Parker, 2000). The Pinus radiata genome has two homologous genes, NEEDLY and PRFLL (Mellerowicz et al., 1998; Mouradov et al., 1998). NEEDLY was shown to be expressed during vegetative and reproductive development, while PRFLL was expressed preferentially in male cones during reproductive development. A search for LFY homologues in basal angiosperms and Gnetales showed that duplicated LFY homologues may be present (Frohlich and Meyerowitz, 1997). Frohlich further proposed the ‘Mostly Male’ theory based on the loss of one of the LFY homologues by the ancestors of the angiosperm lineage (Frohlich and Parker, 2000). Nevertheless, more recently two LFY copies have been reported to occur in some angiosperm species such as tobacco (Ahearn et al., 2001), apple (Wada et al., 2002), and maize (Bomblies et al., 2003). In addition, the phylogenetic tree shown in Fig. 3 clearly showed that the duplication event that gave rise to the apple FLO/LFY homologue occurred before the divergence of the Maloideae.
Northern and in situ hybridization experiments analysis showed that HbLFY expression was restricted to the reproductive tissues, and that HbLFY transcripts accumulated in the floral buds and floral organs, and in the vegetative-to-reproductive transition apex (Fig. 4). Despite the fact that H. brasiliensis has male, female, and bisexual flowers that can be recognized either by their morphology or their predicted location within the inflorescence, all floral meristems expressed HbLFY transcripts equally. The distinction between male and female inflorescences in maize would apparently require the differential expression of distinct maize FLO/LFY paralogues (Bomblies et al., 2003) and a similar suggestion has been presented by Rottmann et al. (2000), driven by the observation that one Populus FLO/LFY homologue promoted the ‘masculinization’ of female Populus trees when over-expressed in transgenic plants.
In addition, in other woody species where FLO/LFY homologues have been cloned, the expression patterns are not always related to the reproductive development. In Eucalyptus and grape, the expression of the corresponding FLO/LFY homologues was also observed in leaf primordia (Dornelas et al., 2004; Southerton et al., 1998). If HbLFY is not expressed in vegetative tissues nor in the vegetative meristem, then there must be an alternative mechanism that regulates its expression according to the season. Seasonal alternating expression of FLO/LFY homologues has also been reported for other woody species such as apple, kiwifruit, and grape (Walton et al., 2001; Carmona et al., 2002; Wada et al., 2002). In these species, the expression levels of their FLO/LFY homologues increase in the proliferating inflorescence meristems generating inflorescence branches, with the highest levels being detected in young floral meristems (Walton et al., 2001; Carmona et al., 2002). Therefore, as observed in grapevine and kiwifruit, the highest levels of HbLFY expression correspond to the time of flower meristem formation, supporting a role for the HbLFY gene product in this process. HbLFY is also expressed in floral organ primordia, and its expression declines as organs expand, as described for other species (Weigel et al., 1992; Hofer et al., 1997; Southerton et al., 1998). In summary, expression of HbLFY in reproductive meristems and developing floral organs suggests that HbLFY plays an important role during the rubber tree reproductive development, as has been suggested for most FLO/LFY-like genes studied.
Aimed at a better understanding of the biological role of HbLFY during reproductive development, transgenic Arabidopsis lfy mutant lines were obtained that carried a LFY::HbLFY construct (Fig. 5). The transgenic (CAPs genotyped) lines were observed for 50–60 d under long-day conditions, and all lines showed a wild-type phenotype. It suggests that HbLFY worked as a functional orthologue of LFY in Arabidopsis plants and its expression under the LFY promoter complemented the lfy-26 phenotype (Weigel and Nilsson, 1995). There are some reports available that describe transgenic Arabidopsis plants expressing LFY homologues from other plant species (Kyozuka et al., 1998; Mouradov et al., 1998; Southerton et al., 1998; Rottman et al., 2000; Shindo et al., 2001; Ahearn et al., 2001; Wada et al., 2002; Dornelas et al., 2004). In all these cases, proteins were over-expressed under the control of the 35S promoter, in either a lfy-26 mutant background (Mouradov et al., 1998; Shindo et al., 2001); or in the wild-type background (all other cases). The exceptions are the expression of NLY at physiological levels in the mutant using the promoter sequence from Arabidopsis LFY (Mouradov et al., 1998; Dornelas et al., 2004), using a similar approach to the one described here. This is the best approach available to assess conservation of function between homologous genes in the absence of homologous recombination. Arabidopsis plants carrying transgenic lines over-expressing NLY from radiata pine and ELF1 from Eucalyptus showed early flowering and produced solitary flowers from rosette axils and terminal flowers from primary shoots. Despite the phylogenetic origin of the transgenic FLO/LFY homologues (from a gymno- or an angiosperm), and the fact that NLY is preferentially expressed in female cones, the activity of both homologues seemed to be very similar in the final phenotype of Arabidopsis transgenic plants. Likewise, despite the fact that HbLFY is expressed in mono- and bisexual flowers in H. brasiliensis, its activity was sufficient to restore the normal development of Arabidopsis bisexual flowers. It also indicates that the separation of sex types within the rubber tree inflorescence may not require a FLO/LFY homologue or may use a molecular pathway that is not conserved in Arabidopsis.
Although the available information suggests that over-expression of LFY is sufficient to promote the conversion of shoots into flowers in woody species such as Populus spp. (Weigel and Nilsson, 1995) and Citrus spp. (Peña et al., 2001), the role of the endogenous FLO/LFY homologues and their function during meristem development are poorly understood in woody species. It is hoped that with the use of reverse genetics both in herbaceous and woody model plants it will be possible to characterize the role of HbLFY during rubber tree reproductive development further.
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Acknowledgements |
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We thank FCA Tavares and G Bandel for providing excellent research environment, D Weigel for the generous gifts of plasmids pDW124, pDW132, and pSKI015, TAIR and the Ohio State University for the Arabidopsis seed stock maintenance, EW Kitajima, for maintaining the scanning electron microscope facility at NAP/MEPA (University of São Paulo, ESALQ, Piracicaba, Brazil), and FAPESP, CAPES, and CNPq for financial support.
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Footnotes |
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* Accession numbers: AY639378 (HbLFY gene) and AY639379 (HbLFY mRNA).
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References |
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