Identifying Eucalyptus expressed sequence tags related to Arabidopsis flowering-time pathway genes

Identificação de etiquetas de seqüências expressas (ESTs) de Eucalytpus relacionadas a genes das vias controladoras do florescimento de Arabidopsis

RESESARCH ARTICLE

Identifying Eucalyptus expressed sequence tags related to Arabidopsis flowering-time pathway genes

Identificação de etiquetas de seqüências expressas (ESTs) de Eucalytpus relacionadas a genes das vias controladoras do florescimento de Arabidopsis

Marcelo Carnier Dornelas^I,II,*; Adriana Pinheiro Martinelli Rodriguez^I

^IUniversidade de São Paulo, Centro de Energia Nuclear na Agricultura, Laboratório de Biotecnologia Vegetal

IIPresent address: Universidade Estadual de Campinas, Instituto de Biologia, Departamento de Fisiologia Vegetal, CP 6109, Cidade Universitária ¨Zeferino Vaz¨, CEP 13084-971 Campinas, SP, Brasil

ABSTRACT

Flowering initiation depends on the balanced expression of a complex network of genes that is regulated by both endogenous and environmental factors. The timing of the initiation of flowering is crucial for the reproductive success of plants; therefore, they have developed conserved molecular mechanisms to integrate both environmental and endogenous cues to regulate flowering time precisely. Extensive advances in plant biology are possible now that the complete genome sequences of flowering plants is available and plant genomes can be comprehensively compared. Thus, association studies are emerging as powerful tools for the functional identification of genes involved on the regulation of flowering pathways. In this paper we report the results of our search in the Eucalyptus Genome Sequencing Project Consortium (FORESTS) database for expressed sequence tags (ESTs) showing sequence homology with known elements of flowering-time pathways. We have searched the 33,080 sequence clusters in the FORESTS database and identified Eucalyptus sequences that codify putative conserved elements of the autonomous, vernalization-, photoperiod response- and gibberellic acid-controlled flowering-time pathways. Additionally, we have characterized in silico ten putative members of the Eucalyptus homologs to the Arabidopsis CONSTANS family of transcription factors.

Keywords: CONSTANS, data mining, EST, photoperiod, vernalization.

RESUMO

As decisões envolvidas na iniciação do processo de florescimento dependem da expressão equilibrada de uma rede complexa de genes, que é regulada por fatores endógenos e ambientais. A regulação correta da transição para o florescimento é crucial para o sucesso reprodutivo das plantas; dessa forma elas desenvolveram mecanismos moleculares conservados para integrar tanto indícios ambientais como endógenos para regular precisamente o tempo do florescimento. Avanços recentes na biologia molecular vegetal, incluindo o seqüenciamento completo de genomas, tornaram possível a comparação detalhada de genomas vegetais. Assim, estudos comparativos de genômica funcional estão emergindo como ferramentas poderosas para a identificação de genes envolvidos no regulamento das vias metabólicas relacionadas ao florescimento. Neste artigo são apresentados resultados da busca no banco de dados do Projeto FORESTS (Eucalyptus Genome Sequencing Project Consortium) para a obtenção de etiquetas de seqüências expressas (ESTs) mostrando homologia com elementos-chave das vias reguladoras do florescimento. Os 33.080 ¨clusters¨ de ESTs de Eucalyptus spp. disponíveis no banco de dados do FORESTS foram analisados, e identificaram-se elementos conservados, possivelmente envolvidos nas vias controladoras do florescimento, quer sejam as autônomas, quer as controladas por vernalização, por ácido giberélico ou por fotoperíodo. Adicionalmente, foram caracterizados in silico dez possíveis membros da família CONSTANS de fatores de transcrição de Arabidopsis, em Eucalyptus.

Palavras-chave: CONSTANS, EST, fotoperíodo, mineração de dados, vernalização.

INTRODUCTION

Flowering is controlled by environmental conditions and developmental regulation (Reeves and Coupland, 2000; Samach and Coupland, 2000; Araki, 2001). The complexity of this regulation is created by an intricate network of signaling pathways (Reeves and Coupland, 2000; Samach and Coupland, 2000; Araki, 2001; Mouradov et al., 2002). Studies in the model plant Arabidopsis have led to the identification of components within individual signaling pathways that affect flowering, and to their positioning within molecular hierarchies. Arabidopsis is an excellent model system in which to approach this complexity, because it responds to many of the environmental conditions that control flowering in other species, and for which genetic tools are well developed (Levy and Dean, 1998; Simpson and Dean, 2002). Furthermore, distinct signaling pathways are known to converge on the activation of the same flowering-time genes (Mouradov et al., 2002; Simpson and Dean, 2002). This convergence of pathways on a common set of genes may enable the integration of different responses, so that the plant can produce a coordinated flowering process under conditions in which multiple environmental parameters are changing simultaneously (Levy and Dean, 1998; Simpson and Dean, 2002; Izawa et al., 2003). Also, genetic analysis of Arabidopsis varieties showing natural variation in flowering time has demonstrated how the activity of these pathways can be altered in nature and how balancing the effects of different environmental stimuli on flowering time is important in plants adapting to growth in different geographical locations (Koornneef et al., 1994; Samach and Coupland, 2000; Araki, 2001).

At present, the full complexity of the flowering network can only be approached in Arabidopsis and probably rice, where the necessary tools are available (Izawa et al., 2003), nevertheless extensive efforts are being made to describe related pathways in other plant species (Mouradov et al., 2002; Izawa et al., 2003; Dornelas and Rodriguez, 2001 and 2004; Dornelas et al., 2004;). Additionally, there is a need to understand how the full diversity in flowering responses is generated. For example, Arabidopsis responds to photoperiod, but all ecotypes are long-day plants that flower earlier under long than short days, whereas many other species show the reverse response and many are not responsive to photoperiod at all (Mouradov et al., 2002; Izawa et al., 2003). Also, all Arabidopsis ecotypes are annual plants, and understanding the perennial habit will require a different model species. Thus, to understand the diversity in flowering responses, there is a need to search for conserved key elements on the regulatory pathways involved in the control of flowering time in other plant species.

We are interested in characterizing genes involved in the early stages of floral development in woody angiosperm trees of the genus Eucalyptus. With this aim, we have used the sequences of the key proteins of the different developmental pathways involved in the regulation of flowering-time available from Arabidopsis as baits to search the Eucalyptus Genome Sequencing Project Consortium (FORESTS) database for expressed sequence tags (ESTs) showing sequence homology with known elements of flowering-time pathways. We have identified Eucalyptus sequences that codify putative conserved elements of the vernalization, photoperiod response, autonomous and GA-controlled flowering-time pathways. Additionally, we have undertaken an extensive in silico characterization of the putative Eucalyptus homologues of the CONSTANS gene family, which, in Arabidopsis, mediate the cross-talk between the circadian clock and the genes controlling reproductive meristem identity. The Arabidopsis CO-like (COL) gene family is proposed to encode proteins with two zinc fingers loosely related to those of GATA transcription factors, termed the B-Box (Putterill et al., 1995) and contains a carboxy-terminal domain called CCT because it is present in CO, COL, and TIMING OF CAB 1 (TOC1) proteins (Strayer et al., 2000; Robson et al., 2001). We expect that our results will contribute to further studies describing how these pathways function in species that respond differently to environmental conditions than does Arabidopsis.

MATERIAL AND METHODS

Searching Eucalyptus ESTs homologs to Arabidopsis flowering-time genes: The overall goal of this study was to retrieve from the FORESTS data set, Eucalyptus spp homologs to all genes described to be involved in the control of flowering time, according to the processes showed in figure 1. In order to achieve this, data mining in the FORESTS database was carried out using published plant gene sequences as baits, as well as keyword searches in the FORESTS home page (https://forests.esalq.usp.br). Plant gene sequences used as baits were retrieved from public gene databases (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi) using their corresponding accession numbers or by the use of keyword-oriented searches (Mouradov et al., 2002; Izawa et al., 2003). Protein (deduced amino acid) sequences from the retrieved bait sequences were compared to Eucalyptus clustered EST sequences using tBLASTn or a combination of different Blast algorithms (Altshul et al., 1997), with the BLOSUM62 scoring matrix, with a threshold of e<10^-10 for positive hits. The identity (in terms of donor cDNA library) and number of sequence read composition of each individual candidate cluster were checked to access their potential expression pattern.

For the results presented in table 1, we have obtained e-values using the BLASTp algorithm (Altshul et al., 1997) as described above and calculated the identity and the similarity, at the amino acid level, relative to the corresponding Arabidopsis putative homolog, within the extension of the successful sequence alignment produced by the pair-wise comparison.

In silico characterization of the Eucalyptus homologs belonging to the CONSTANS gene family: The Arabidopsis CONSTANS (CO) gene family codifies putative transcription factors defined by two conserved domains (Putterill et al., 1995; Griffiths et al., 2001). The first is a zinc finger region near the amino terminus that resembles B-boxes, which regulate protein-protein interactions in several animal transcription factors (Putterill et al., 1995). The second is a region of 43 amino acids near the carboxy terminus termed the CCT (CO, CO-like, TOC1) domain (Robson et al., 2001). We have identified Eucalyptus homologs to the Arabidopsis CO gene family, using the Arabidopsis sequences as baits and the BLAST algorithms (Altshul et al., 1997) as described above. Only comparisons that produced an e-value lower than e^-50 were considered highly significant. When the obtained e-values were between e^-50 and e^-5, a re-clusterization of all reads identified was performed using the CAP3 algorithm from the BioEdit Software (Hall, 1999). The novel cluster consensus sequences obtained were re-submitted to BLAST and frequently better e-values were obtained. To identify the presence of conserved domains in the deduced protein sequence, we analyzed them using the CDD algorithm (Marchler-Bauer et al., 2005).

Comparative and phylogenetic analysis of CONSTANS gene family homologs: To examine the relationships between the Eucalyptus CO-like genes and their putative Arabidopsis homologs in more detail, their nucleotide and predicted peptide sequences were used to determine genetic distances and to construct phylogenetic trees. Because the middle regions of the genes were the most diverged, they could not be aligned with confidence. Therefore, neighbor-joining (NJ) and maximum parsimony (MP) trees were constructed using B-box (and CCT domain sequences when available) following the alignments obtained using the CLUSTALX software (Thompson et al., 1994). The alignments were eventually corrected by hand. Phylogenetic trees were obtained using parsimony and/or genetic distance calculations. Neighbour-joining (Saitou and Nei, 1987) and Bootstrap (with 1000 replicates) trees were built using the MEGA software (http://www.megasoftware.net).

RESULTS

Eucalyptus ESTs homologs to Arabidopsis flowering-time genes: Based on the systematic search in the FORESTS database using Arabidopsis sequences as baits, we have identified 536 Eucalyptus spp. EST sequences, organized in 21 clusters, representing putative Eucalyptus homologs to flowering-time genes. Some of these genes are required for the daylength response, and some encode regulatory proteins specifically involved in the control of flowering, while others encode components of light signal transduction pathways or are involved in circadian clock function. A representation of the relationships among these processes is shown in figure 1 and the putative homologs of the key players in Eucalyptus are presented in table 1. The role of each of these elements in the flowering-time pathways and their implication for the understanding of the Eucalyptus flowering processes are presented in the Discussion section.

Comparative and phylogenetic analysis of Eucalyptus CONSTANS-like gene family: We have found 187 Eucalyptus EST sequences showing significant (e-value lower than e^-5) similarity to the Arabidopsis COL genes, by means of a combination of the tBLASTn and keyword search in the FORESTS database. When submitted to the CAP3 algorithm, these sequences were organized initially in 37 clusters. Their deduced amino acid sequences were submitted to BLASTp and the number of clusters showing significant (e-value lower than e^-5) similarity to the Arabidopsis COL genes was reduced to 20.

We thus restricted our analysis to Eucalyptus sequences showing the conserved B-box and CCT domains, according to the definition of the COL family provided by Griffiths et al., (2003). The number of Eucalyptus EST clusters encoding proteins significantly similar to those from the Arabidopsis COL family was then reduced to ten. We thus assumed that each of these clusters corresponds to a member of the Eucalyptus COL gene family (table 2).

As most of the CCT domain sequences are not available for the EgCOL proteins, we produced alignments of the predicted peptides of the conserved B-box region for all Arabidopsis AtCO and AtCOL proteins and their putative Eucalyptus homologs (figure 2A). The alignment showed that the Eucalyptus EgCO and EgCOL2-10 had consensus CO-like amino acid residues at the amino-terminus. Variation within the B-box domain suggested that the CO-like genes could be further subdivided. For example, distinctive amino acid residues in the B-boxes grouped Arabidopsis COL3 to COL5 peptides with EgCOL2 and EgCOL3 (figure 2A).

To further examine the relationship between the putative EgCOL homologs and their Arabidopsis counterparts, the sequence alignment shown in figure 2A was used to determine genetic distances and to construct phylogenetic trees. Therefore, neighbor-joining (figure 2B) and maximum parsimony trees were constructed, giving similar results (data not shown). This consistently grouped the proteins into three main clades (figure 2B). Group III genes comprised Arabidopsis and Eucalyptus proteins with two zinc finger domains, the second of which was diverged from the CO-type B-box. Group II genes comprised Arabidopsis and Eucalyptus proteins with a single B-box. Group I comprised the majority of CO-like genes and included the Eucalyptus EGCO, EgCOL2 and EgCOL3 proteins. There were subdivisions within Group I, but these had low bootstrap values (figure 2B). EgCOL7 had the most diverged B-Box domain and the phylogenetic tree placed it, and the related Arabidopsis proteins ATCOL16 and AtCOL6-7, within Group II. Other two Eucalyptus proteins, EgCOL5 and EgCOL6 were consistently divergent and were also placed in Group II, at the base of the tree, but in a different clade to which no Arabidopsis homologs could be assigned.

DISCUSSION

The apical meristem in most Eucalyptus species generally remains vegetative throughout their life cycle. Lateral meristems, arising in the leaf axils, may give rise to a leafy shoot or to an inflorescence (Dornelas et al., 2004), in response to inductive environmental conditions such as day length and temperature, as well concentrations of endogenous substances such as gibberellic acid (GA), if the tree is sufficiently mature (Moncur and Hasan, 1994; Southerton et al., 1998). Molecular and genetic approaches have identified genes required for the flowering-time control and their relationships were established in the model plant Arabidopsis (figure1). We have identified putative homologs of the key players in Eucalyptus (table 1). Additionally, we have characterized in silico, the putative Eucalyptus homologs to the COL gene family. In the following sections we discuss the role of each of these elements in the flowering-time pathways and their implication for the understanding of the Eucalyptus flowering process.

The photoperiod response pathway and the role of the CONSTANS gene family

One of the most important environmental factors controlling flowering time is the duration of the daily light period, or photoperiod. Arabidopsis is a facultative long-day plant, which flowers earlier under long days but eventually flowers under short days (Corbesier et al., 1996). The products of the genes CONSTANS (CO), CRYPTOCHROME2/FHA (CRY2), GIGANTEA (GI), and FT are part of this long day–promoting pathway (Koornneef et al., 1991; Mouradov et al., 2002). CO is the latest acting of the known genes that is specific to this pathway; all the other genes also act in other pathways or have more general effects (figure 1). FT acts downstream of CO and also in other pathways (Kardailsky et al., 1999; Kobayashi et al., 1999; Onouchi et al., 2000), whereas GI and CRY2 act upstream of CO (Guo et al., 1998; Suarez-Lopez et al., 2001).

The CO gene was proposed to encode a protein with two zinc fingers loosely related to those of GATA transcription factors (Putterill et al., 1995) and contains a carboxy-terminal domain called CCT because it is present in CO, CO-like (COL), and TIMING OF CAB 1 (TOC1) proteins (Strayer et al., 2000; Robson et al., 2001). Comparison of 16 Arabidopsis COL genes that encode proteins sharing the B-box and CCT domains (Robson et al., 2001) demonstrated that the B-box is a type of zinc finger identified in animal proteins and believed to mediate protein–protein interactions (Borden, 1998). In an analysis of the Arabidopsis CO-like genes sequences by Lagercrantz and Axelsson (2000), they concluded that this gene family evolves rapidly, particularly in the middle regions. Their analysis focused on B-box sequences only, and they included the Arabidopsis STO (SALT TOLERANCE) gene. STO-like genes have B-boxes but no CCT domain. Additionally, their analysis included in the COL family, the related ZIM gene, which contains an additional ZIM motif. This short motif is found in a variety of plant transcription factors that contain GATA domains and its conserved amino acids form the pattern TIFF/YXG (Lagercrantz and Axelsson, 2000; Griffiths et al., 2003).

We have identified ten members of the COL gene family in Eucalyptus (table 2; figure 2). Each member was represented by a single EST sequence (i.e. a singleton, e.g. EgCOL10) or by the re-clusterization of more than one cluster, represented by more than 40 sequences (e.g. EgCOL2). Among the sequences that composed the clusters, we found many from cDNA libraries derived from tissues of E. camaldulensis, E. urophylla and E. saligna, additionally to those derived from E. grandis. We could identify COL members coding for all three conserved groups of COL proteins (figure 2B). These three groups were identified previously and are thought to have evolved prior to the divergence of monocots and dicots (Griffiths et al., 2003). COL proteins belonging to different groups are expected to perform distinct biological roles, although only the CO role in controlling flowering time has been studied in detail (Onouchi et al., 2000; Suarez-Lopez et al., 2001; Griffith et al., 2003).

When constitutively expressed in Arabidopsis, CO induces early flowering and loss of photoperiod sensitivity (Onouchi et al., 2000). Accordingly, mutations in the FT, SUPPRESSOR OF OVEREXPRESSION OF CO1 (SOC1, also called AGAMOUS-LIKE 20 [AGL20]) genes partially suppress the early flowering phenotype of 35S::CO (Onouchi et al., 2000).

The CO transcript level shows a diurnal peak in Arabidopsis plants grown in long days. This peak is narrower in short days, ending 4 h earlier (Suarez-Lopez et al., 2001). Plants entrained in long days show a circadian rhythm in CO transcript level when transferred to continuous light (LL), indicating that this rhythm is controlled by the circadian clock (Suarez-Lopez et al., 2001). FT is an early target of CO (Samach et al., 2000), and its transcript level also follows a circadian rhythm (Suarez-Lopez et al., 2001). This peak is absent in the co mutant. In addition, the circadian clock–related mutants, LATE ELONGATED HYPOCOTYL (LHY), GI, and EARLY FLOWERING3 (ELF3), show an altered rhythm in CO transcript level that correlates with their flowering phenotype. Overall, CO appears to mediate between the circadian clock and the flowering-time gene FT (Suarez-Lopez et al., 2001). Candidates to photoreceptors that might be involved in the post-transcriptional activation of CO protein under long days are CRY2 and PHYTOCHROME A (PHYA).

The circadian clock elements

The molecular mechanism that generates the circadian rhythms is often described as being composed of three interrelated parts. The first one is an input pathway that synchronize the clock mechanism to daily cycles of light and dark; the second one is a central oscillator that generates the 24-h time-keeping mechanism; and the third one is a group of output pathways that regulate particular processes (Roenneberg and Merrow, 2000; McClung, 2001). The control of flowering via CO and FT represents one such output pathway (Suarez-Lopez et al., 2001). Another output pathway, represented by the CHLOROPHYLL A/B BINDING PROTEIN2 (CAB2) gene, has enabled detailed analysis of clock regulation. The Arabidopsis timing of CAB (toc) mutants flowered early in short days (Kreps and Simon, 1997; Somers et al., 1998). The TOC1 protein shows two characteristic domains (Strayer et al., 2000). The first of these, at the N terminus, is similar to the receiver domain of response regulators from two-component signal transduction systems. The second motif, at the C terminus, is the CCT domain, shared with the CO family of transcriptional regulators. The consensus cluster sequence encoding the putative Eucalyptus TOC1 homolog was formed by an alignment of sequences derived from cDNA libraries made with E. grandis, E. saligna and E. urophylla tissues, indicating that TOC homologs may be conserved among Eucalyptus species.

Other likely components of the oscillator are the CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) genes, which are also involved in the photoperiodic induction of flowering. These genes encode highly similar single Myb domain DNA binding proteins and are each regulated by the circadian clock, with a peak in their expression soon after dawn (Schaffer et al., 1998; Wang and Tobin, 1998). The overexpression of LHY or CCA1 in Arabidopsis disrupts the circadian rhythm and causes late flowering under long-day conditions (Schaffer et al., 1998; Wang and Tobin, 1998; Mizoguchi et al., 2002). Recent reports, nevertheless, suggest that LHY overexpression may not disrupt rhythms in expression of the circadian clock–regulated gene EARLY FLOWERING 3 (ELF3), which would be an unexpected exception to the effect of LHY (Hicks et al., 2001). ELF3 encodes a nuclear protein proposed to act as a transcriptional activator (Hicks et al., 2001; Liu et al., 2001).

According to the current molecular model (figure 1), LHY and CCA1 feed back to repress their own expression, and as their protein levels fall, the expression of TOC1 rises. TOC1, in turn, promotes the expression of LHY and CCA1, so initiating another cycle (Alabadi et al., 2001). All central elements of the model have been recently described to occur in rice (Izawa et al., 2003) and have shown to have putative homologs in Eucalyptus (table 1), suggesting the conservation of the molecular mechanism regulating the circadian clock in angiosperms

Synchronizing the molecular circadian clock

Circadian rhythms must be synchronized with the daily rhythm of light and dark and of temperature (Roennenberg and Merrow, 2000). The phase of circadian rhythms can therefore be reset by environmental conditions such as light and temperature that fluctuate during day/night cycles. The photoreceptors PHYA, PHYB, CRY1, and CRY2 all influence clock entrainment under specific light conditions (McClung, 2001; Yanovsky et al., 2000). Mutations in an Arabidopsis PHYB-interacting protein, ZEITLUPE (ZTL), also have dramatic effects on clock function and flowering time (Somers et al., 2000). Constitutive overexpression of ZTL gave a late-flowering phenotype in Arabidopsis plants growing in long-days (Nelson et al., 2000). The ZTL protein contains a PAS domain, an F-box, and six repeated kelch motifs (Adams et al., 2000). The PAS domain mediates protein–protein interactions and has been found in a group of blue light photoreceptors (Briggs and Huala, 1999). Accordingly, protein–protein interactions have been described between ZTL and PHYB and between ZTL and CRY1 (Jarillo et al., 2001). ZTL is a member of a family of three genes in Arabidopsis that also includes FKF1 and LKP2 (Somers et al., 2000). Putative homologs for all three genes have been found in Eucalyptus (table 1.). The fkf1 mutant shares the late-flowering phenotype of ztl. The late-flowering phenotype of fkf1 can be rescued by vernalization and gibberellin (GA) application, suggesting that fkf1 may be associated with the autonomous flowering pathway (Nelson et al., 2000).

Mutant alleles of the Arabidopsis gene GIGANTEA (GI) also cause alterations in period length of gene expression rhythms. The GI protein contains six putative transmembrane domains (Fowler et al., 1999; Park et al., 1999; Huq et al., 2000), suggesting a membrane protein, but GUS:GI and GFP:GI fusion proteins were targeted to the nucleus (Park et al., 1999). Three Eucalyptus EST clusters span the GI gene, but the one containing the most extensive 5' region is EGBMST2013E01 (table 1).

The vernalization response pathway

Exposure to low temperatures for several weeks will often accelerate flowering in Arabidopsis (Michaels and Amasino, 2000). The genetic control of vernalization was related to two different loci, FLOWERING LOCUS C (FLC) and FRIGIDA (FRI; Burn et al., 1993; Lee and Amasino, 1993; Clarke and Dean, 1994). FLC encodes a MADS box transcription factor (Sheldon et al., 1999; Michaels and Amasino, 2000) that is a repressor of flowering, and high-level expression of FLC correlates with the vernalization requirement of winter annual Arabidopsis varieties (Michaels and Amasino, 2000; Sheldon et al., 1999; 2000). The other locus at which dominant alleles confer a vernalization requirement is FRI. The product of this gene somehow increases FLC mRNA abundance (Sheldon et al., 1999; Michaels and Amasino, 2000). The biochemical function of FRI protein is unknown, but it contains coiled-coil domains that may be involved in protein–protein interactions (Johanson et al., 2000). FRI homologs were not clearly identified in rice (Izawa et al., 2003). Further, no orthologues of the recently identified VERNALIZATION1 (VRN1) and VRN2 (Gendall et al., 2001; Levy et al., 2002) have been found in rice (Izawa et al., 2003), suggesting that vernalization-related genes have been lost from the rice genome during evolution, probably due to the adaptation to tropical regions. Similarly, no Eucalyptus ESTs were found that showed significant similarities to FRI, VRN1 or VRN2.

Cloning of VRN2 showed that its protein sequence is related to a Drosophila Polycomb group protein, Su(z)12, that regulates gene expression by modifying chromatin structure (Birve et al., 2001; Gendall et al., 2001). This suggests that there could be some molecular mechanism relating chromatin structure and flowering time control.

The relation between chromatin structure and flowering control

Arabidopsis mutants have been described that flower without forming any adult leaves, advancing directly from embryonic development to flowering (Sung et al., 1992; Yang et al., 1995; Kinoshita et al., 2001). The class of mutants named embryonic flower (emf1; Sung et al., 1992 and emf2 Yang et al., 1995) form a reduced inflorescence and abnormal flowers that lack petals without first forming any rosette (Sung et al., 1992; Yang et al., 1995). The predicted EMF2 protein contains a zinc finger, an N-terminal basic domain and a C-terminal acidic domain (Yoshida et al., 2001). This protein belongs to the same family as VRN2 and also showed similarity to the Drosophila Polycomb group protein Su(z)12. The EMF2 protein may then act as part of a protein complex that during embryo and seedling development represses the expression of genes that promote reproductive development. The importance of Polycomb group genes in repressing reproductive development was also emphasized by the demonstration that another Arabidopsis Polycomb group gene, FERTILIZATION INDEPENDENT ENDOSPERM (FIE), is related to the EXTRA SEX COMBS protein of Drosophila (Kinoshita et al., 2001). Loss-of-function fie alleles were originally described because they allow partial endosperm formation prior to fertilization. However, the effect of the mutation in the seedling or adult was not described, because maternal FIE alleles are essential for embryo development (Ohad et al., 1999). By expressing FIE from a defective FIE promoter that allows expression during seed development but not in the germinated seedling, it was demonstrated that FIE is required for the repression of flowering in the seedling (Kinoshita et al., 2001). Plants homozygous for fie and expressing FIE from such a defective promoter initiate reproductive development as seedlings and resemble emf mutants. Putative homologs for both EMF2 and FIE were found among Eucalyptus ESTs in the FORESTS database (table 1).

The GA pathway

The growth regulator gibberellic acid (GA) promotes flowering in Arabidopsis (Wilson et al., 1992; Putterill, et al., 1995; Blazquez et al., 1998). On the other hand, exogenous application of paclobutrazol reduced the concentration of endogenous GA in apical tissues of different Eucalyptus species and enhanced the reproductive activity of grafted trees (Moncur and Hasan, 1994), suggesting that in Eucalyptus, high concentrations of GA inhibits the flowering process. One of the ways in which GAs promote flowering is by increasing the transcriptional activity of the floral meristem identity gene LEAFY (LFY). Overexpression of LFY also restores flowering of ga1-3 in short days (Blazquez et al., 1998). No Eucalyptus EST in the FOREST database showed significant similarity to LFY. Nevertheless, LFY orthologs have been identified in E. grandis (Dornelas et al., 2004) and in E. globulus (Southerton et al., 1998), indicating that the GA-regulated flowering pathway mediated by LFY may be conserved in Eucalyptus.

Integration of flowering pathways

Separate genetic pathways regulate flowering in response to different environmental signals, but these pathways eventually converge to regulate the expression of the same downstream genes. For example, all of the flowering-time pathways ultimately lead to the transcriptional activation of the same set of floral identity genes that act within the floral primordia (Pineiro and Coupland, 1998). LFY is the earliest of the known floral identity genes to be expressed, and directly activates later genes (Wagner et al., 1999). Plants carrying fusions of the LFY promoter to the GUS marker gene were used to demonstrate that LFY expression responds both to the long-day flowering pathway and to GA. Thus the GA and long-day pathways converge on the LFY promoter, rather than both pathways activating an earlier acting gene that in turn increases the expression of LFY (Blazquez and Weigel, 2000). Convergence of the long-day and autonomous pathways has also been studied. These pathways are clearly separate upstream FLC and CO genes (figure 1). Nevertheless, flowering-time genes that act downstream of both CO and FLC have been identified. The SOC1 (or AGL20) gene that encodes a MADS box protein is both activated by CO and repressed by FLC, suggesting that the pathways represented by these genes converge on SOC1 (Borner et al., 2000; Lee et al., 2000; Samach et al., 2000; Michaels and Amasino, 2001). SOC1 is also regulated by GA, and therefore is a common target of all three flowering pathways (Borner et al., 2000). Expression of FT, another flowering-time gene regulated by more than one pathway, is reduced by mutations that impair the function of the long-day and autonomous pathways and activated by overexpression of CO, indicating that FT acts downstream of both of these pathways (Samach et al., 2000). The relationship between FT and floral meristem identity gene expression has been studied at the genetic and molecular levels (Nilsson et al., 1998). FT is a member of a small gene family in Arabidopsis that also contains TERMINAL FLOWER 1 (TFL1). The exact biochemical function of this family of proteins in plants is not known, nevertheless both FT and TFL1 putative homologs were found in Eucalyptus (table1). The large conservation of all key regulators of the flowering-time pathways between Arabidopsis and Eucalyptus suggests that most of the molecular mechanism regulating the flowering process may be evolutionarily conserved among Myrtaceae and Brassicaceae, despite their different flowering strategies.

Conclusions and Perspectives

Genetic analysis in Arabidopsis has enabled the isolation of genes that control flowering time and the identification of interacting pathways that promote flowering in response to different environmental conditions. However, the function of key pathway components is likely to be altered in other herbaceous as well as woody plants to change their target genes or the activity of the protein complexes in which they act. These changes may explain, for example, why GA promotes flowering in Arabidopsis but represses flowering in Eucalyptus and some short-day plants (Moncur and Hasan, 1994; Izawa et al., 2003). Thus, there are flowering strategies for which Arabidopsis may be a less-effective model. Accordingly, although Arabidopsis genes can be used to drive very early flowering of trees and to overcome developmental delays of flowering (Weigel and Nilsson, 1995; Peña et al., 2001), studies of an annual plant such as Arabidopsis will probably not reveal the molecular mechanisms underlying many of the unique features of perennial plants (Battey, 2000). Thus, we hope that the identification of putative homologs of the key genes involved in the control of the transition to flowering in Eucalyptus presented in this article, as well as future studies on their expression patterns and on their manipulated expression in transgenic plants, will enable comparisons of the molecular mechanisms that regulate the floral transition in higher plants.

Acknowledgments: The authors acknowledge FAPESP, CAPES and CNPq for financial support.

Received: 21/05/2005, Accepted: 28/06/2005

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*

Corresponding author:Av. Centenário, 303, CEP 13400-970 Piracicaba, SP, Brasil

Publication Dates

• Publication in this collection
  01 Sept 2005
• Date of issue
  June 2005

History

• Accepted
  28 June 2005
• Received
  21 May 2005