Identification of Soybean Genes Involved in Circadian Clock Mechanism and Photoperiodic Control of Flowering Time by In Silico Analyses

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1 Journal of Integrative Plant Biology 2007, 49 (11): Identification of Soybean Genes Involved in Circadian Clock Mechanism and Photoperiodic Control of Flowering Time by In Silico Analyses Vera Quecini 1, Maria I. Zucchi 1, José Baldin 2 and Natal A. Vello 2 ( 1 Instituto Agronômico de Campinas, Centro de Pesquisa e Desenvolvimento de Recursos Genéticos, Campinas, SP , Brazil; 2 Departamento de Genética, ESALQ, Universidade de São Paulo, Piracicaba, SP , Brazil) Abstract Glycine max is a photoperiodic short-day plant and the practical consequence of the response is latitude and sowing period limitations to commercial crops. Genetic and physiological studies using the model plants Arabidopsis thaliana and rice (Oryza sativa) have uncovered several genes and genetic pathways controlling the process, however information about the corresponding pathways in legumes is scarce. Data mining prediction methodologies, including multiple sequence alignment, phylogenetic analysis, bioinformatics expression and sequence motif pattern identification, were used to identify soybean genes involved in day length perception and photoperiodic flowering induction. We have investigated approximately sequences from open-access databases and have identified all bona fide central oscillator genes and circadian photoreceptors from A. thaliana in soybean sequence databases. We propose a working model for the photoperiodic control of flowering time in G. max, based on the identified key components. These results demonstrate the power of comparative genomics between model systems and crop species to elucidate the several aspects of plant physiology and metabolism. Key words: circadian clock; cryptochrome; data mining; flowering; photoperiod; phytochrome. Quecini V, Zucchi MI, Baldin J, Vello NA (2007). Identification of soybean genes involved in circadian clock mechanism and photoperiodic control of flowering time by in silico analyses. J. Integr. Plant Biol. 49(11), Available online at Life on earth is highly influenced by seasonal changes in the environmental conditions; such as light, temperature and rainfall. Thus, the organisms maximize their chances of survival and reproductive success by adjusting the timing of the crucial developmental processes to the most favorable time of the year (Yanovsky and Kay 2003). Multiple pathways quantitatively regulate a group of common targets, the floral pathway integrators, whose activation converts the shoot apical meristem into a flower meristem. An emerging concept is that the alternation between the predominant stimuli contributes to the plasticity and diversity of the control of the transition to the reproductive stage in plants, due to the antagonistic effects of flower-inducing factors on floral repressors (Boss et al. 2004). The concept is Received 14 Nov Accepted 22 Jun Author for correspondence. Tel: ext. 452; Fax: ; <vquecini@iac.sp.gov.br>. C 2007 Institute of Botany, the Chinese Academy of Sciences doi: /j x in agreement with previous studies in soybean, which showed that the genes controlling the juvenile period function in nonoverlapping parallel pathways to the genes controlling the photoperiodic response (reviewed by Destro et al. 2001). Plants measure the day length and discriminate day from night using a complex system of photoreceptor molecules that integrate environmental light information with the endogenous time keeping mechanism, the circadian clock (Yanovsky and Kay 2003). Circadian clocks synchronize the biological processes in prokaryotic and eukaryotic cells with the environmental diurnal cycles, allowing the organisms to anticipate the periodic changes in the environment (Harmer et al. 2000). Circadian clocks are composed of an input pathway that perceives the environmental entraining conditions such as light and, to a much less extent, temperature; the central oscillator consisting of auto-regulatory transcriptional feedback loops that transmit the pace of the clock to downstream components generating the output pathway, and the overt rhythms (Bell-Pedersen et al. 2005). In soybean, genetic control of flowering time has been used by classical breeding programs for many years and is essential for efficient cropping in different latitudinal and climatic regions

2 In Silico Candidates for Photoperiodism in Soybean 1641 (Curtis et al. 2000), but molecular information about the genes involved has not yet emerged into the public domain. The vast majority of soybean commercial cultivars require short days to induce flowering, whereas the photoperiodic response is repressed under long days. Classic genetic studies have demonstrated that the control of photoperiodic flowering in Glycine max involves eight loci that also affect the length of the juvenile period: E1 and E2 (Bernard 1971), E3 (Buzzell 1971), E4 (Buzzell and Voldeng 1980), E5 (McBlain and Bernard 1987), E6 (Bonato and Vello 1999), E7 (Cober and Voldeng 2001), and J (Ray et al. 1995). In general, late flowering and long juvenile periods are dominant or partially dominant, whereas early flowering and maturity are recessive, except for the locus E6, in which early flowering is dominant. Thus, these loci act similarly to photo-stable phytochromes in Arabidopsis, inhibiting flowering under non-inductive conditions (Mockler et al. 2003). The loci E1, E3, E4 and E7 are responsible for the transition to flowering under artificially created long days (Buzzell 1971; Buzzell and Voldeng 1980; Saindon et al. 1989; Cober et al. 1996; Cober and Voldeng 2001), demonstrating that these alleles have distinct sensibilities to light quality, the ratio between R and FR (R:FR). The locus E3 is insensitive to light quality and delays flowering in long days, regardless of the light quality (Cober et al. 1996), while E1, E4 and E7 require lower R:FR ratios to delay flowering, causing the absence of late flowering under high R irradiations (Cober et al. 1996; Colbert and Voldeng 2001). The recessive allele e3 appears to control long-day insensitivity under fluorescent light in an independent manner (Buzzell 1971), whereas e4 requires the presence of e3 to control long day-insensitivity under incandescent light (Buzzell 1971; Buzzell and Voldeng 1980). The alelle e1 is incapable of inducing long-day insensitivity under incandescent light in the presence of an E4 allele that is probably responsible for long-day insensibility along with e1 (Abe et al. 2003). The present work was set out to investigate the circadian temporal programming and its integrative pathways to physiology and metabolism in soybean, using combined in silico expressed sequence tag (EST) profiling and domain structural data analysis. The results presented here demonstrate that the vast majority of the gene products involved in circadian timing in model species is present in soybean. Moreover, genetic distance and domain structure analyses have uncovered extensive amino acid sequence conservation, providing indications of functional equivalence between Arabidopsis and soybean transcripts and demonstrating the incorporation of in silico analyses to gene discovery in non-model species. Results We have compiled Arabidopsis thaliana and Oryza sativa genes involved in the endogenous time-keeping mechanism and photoperiodic flowering induction and their protein sequences were used to search public access G. max databases in order to retrieve their soybean homologs. The sequences were ranked according to the degree of amino acid sequence conservation to be used in tblastx queries of the Arabidopsis genome. Soybean EST contigs retrieving the respective Arabidopsis original bait sequences were ranked and the top scoring hits or hits ranked low but showing highly conserved regions were analyzed further. This way, we identified 33 soybean EST contigs corresponding to Arabidopsis genes involved in the circadian clock mechanism and photoperiodic flowering control. From the total, 12 EST contigs share sequence homology with components of the input pathway to the clock and its ancillary loops, 10 are homologs to components of the central oscillatory mechanism and 11 function in the photoperiodic control of flowering time. Input pathway genes In plants, photoreceptor phytochrome (phy) and cryptochrome (cry) families play a major role in entraining the endogenous time-keeping mechanism according to environmental signals (Millar 2004). In soybean, homologs of phy and cry photoreceptorfamiliesof Arabidopsis were found; the full-length sequences of phya and phyb, a partial cdna that shares sequence homology to AtPHYE, two distinct ESTs corresponding to Arabidopsis CRY1 and one corresponding to CRY2 (Table 1). Previous sequence analyses suggest that these sequences correspond to functional photoreceptors (Hecht et al. 2005; Quecini et al. 2007). The three-member family of putative photoreceptors ZEITLUPE (ZTL), FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 (FKF1), and LOV-KELCH PROTEIN2 (LKP2) family is represented in soybean by two distinct sequences; one highly similar to ZTL and one sharing high sequence similarity to FKF1 and LKP2 (Table 1). Arabidopsis ELF3 is represented by three EST contigs in soybean databases, although with regular homology levels (Table 1). Soybean ELF3 homologs share high modular sequence conservation with its Arabidopsis counterpart throughout the protein (Figure 1A). However, at this point, no functional protein domain has been characterized in ELF3, thus, restricting the bioinformatics power to predict the biological function of soybean homologs. The nuclear protein GIGANTEA (GI) from Arabidopsis is thought to function in an ancillary loop of the input pathway, close to the central oscillator (Mizoguchi et al. 2005) and it is represented by two cdnas in soybean (Table 1). The recognizable motifs at the N-terminal of GI, namely a cysteine dioxygenase type I domain and a conserved sequence of unknown function (DUF561) are virtually absent from soybean partial sequences, however, the homology at the carboxy-terminus of the protein is striking (Figure 1B). This region corresponds to a consensus sequence found in gas vesicle proteins from aquatic organisms, but its biological role in terrestrial plants is uncertain.

3 1642 Journal of Integrative Plant Biology Vol. 49 No Table 1. Glycine max expressed sequence tags (ESTs) with homology to genes of the input pathway to the circadian clock from Arabidopsis thaliana Arabidopsis thaliana Glycine max Protein motifs and References biological process Name Gene EST % e value Photoreceptors CRY1 AT4G08920 BM (P: 80%) 2e B photoreceptor, FAD binding Cashmore et al domain, DNA photolyase BQ (P: 86%) 6e CRY2 AT1G04400 BU (P: 87%) 2e-87 Cryptochrome family Guo et al PHYA AT1G09570 L (FL) 0.0 R/FR photoreceptor, photoreceptor, Sharrock and Quail 1989 PAS1, PAS2, chromophore binding domain, HKL domain (SOYPHYA - P42500) PHYB AT2G18790 L (FL) 0.0 Phytochrome family Boylan et al (SOYPHYB P42499) FKF1 AT1G68050 BQ (P: 21%) 4e-64 Zeitlupe family Nelson et al LKP2 AT2G18915 BQ (P: 29%) 3e-68 Zeitlupe family Schultz et al ZTL AT5G57360 BM (P: 46%) 1e-109 Putative photoreceptor Kelch repeats, F-box domain, LOV domain Somers et al Ancillary factors ELF3 AT2G25930 BE (P: 18%) 2e No recognizable domain, input McWatters et al pathway to circadian clock, gating mechanism BE (P: 19%) 2e TC (P: 15%) 6e GI AT1G22770 BM (P: 35%) 1e Similar to gas vesicle protein; Koornneef et al cysteine dioxygenase type I BG (P: 34%) 2e PIL6 AT3G59060 CA (P: 29%) 7e-174 Transcriptional regulator, MYC-related bhlh domain Fujimori et al Gene name abbreviations: CRY, cryptochrome; ELF, early flowering; FKF, F-box Kelch repeat flavin binding protein; GI, gigantea; LKP, LOV domain, Kelch repeat protein; PFT, phytochrome and flowering time; PHY, phytochrome; PIL, phytochrome-interacting protein-like; SRR, sensibility to red reduced; ZTL, Zeitlupe. Identity percentage at the amino acid level: P, partial coverage percentage; FL, full length clone. Functional domains abbreviations: ARNT, aryl hydrocarbon receptor nuclear translocator; B, blue light; FAD, flavina adenosine dinucleotide; HKL, histidine kinase-like; LOV, light, oxygen, voltage subtype PAS domain; PAS, Per, ARNT, Sym domain. Another nuclear protein is hypothesized to bridge light perception and the circadian clock in Arabidopsis, the nucleuslocalized PFT1 (Cerdán and Chory 2003). It is characterized by a predicted von Willebrand factor type A (vwf-a) domain at its amino terminus and a glutamine-rich region at the carboxy terminus, reminiscent of some transcriptional activators (Cerdán and Chory 2003). The soybean genome has one homolog highly similar at the vwf-a domain but lacking the glutamine-rich region (Figure 1C). In several organisms, vwf-a domains are involved in many cellular processes, and a large number of them show a divalent cation-binding site that in some cases has been shown to mediate protein-protein interactions. In Arabidopsis and soybean, the DxSxS motif responsible for divalent cation binding is converted to ExTxA, although it is unclear whether the partial sequence conservation still allows ion association. We have identified a soybean cdna with sequence homology to the bhlh transcription factor PIL6 (PIF3-LIKE6), it is highly similar to AtPIL6 at the DNA-binding domain and the adjacent plant-specific consensus of unknown function (DUF822) (Figure 1D). The soybean sequence also shares sequence

4 In Silico Candidates for Photoperiodism in Soybean 1643 Figure 1. Sequence alignment of input pathway components of Glycine max and Arabidopsis thaliana circadian clock. (A) ELF3. (B) GI. (C) PFT1 von Willebrant factor type A (vwf-a) domain. (D) PIL6 basic helix-loop-helix (bhlh) domain. Soybean deduced amino acid and Arabidopsis sequences aligned with ClustalX are shown. Dark gray and light gray shaded boxes represent sequence identity and similarity, respectively. similarities with other proteins from the PIL family (Quecini et al. 2007). Central oscillator feedback loop genes All components hypothesized to constitute the translational feedback loop mechanism of the central oscillator in Arabidopsis are present in soybean genome (Table 2). The family of pseudoresponse regulators TOC1/PRR consists of three members in soybean: one with higher sequence similarity to PRR3 and PRR7, another more closely resembling PRR5 and PRR9, and a third one, similar to PRR1/TOC1 (Figure 2A,B). Thus, G. max and Arabidopsis PRR gene family appear to have undergone a differential expansion process during evolution (Figure 2B). Soybean homologs contain the signature pseudo responseregulator receiver domain at the amino terminus region and the consensus 43 amino acids constituting the CONSTANS, CO-like, and TOC1 (CCT domain) (Figure 2A). PRR-like reads were found in reproductive and vegetative tissue libraries from

5 1644 Journal of Integrative Plant Biology Vol. 49 No Table 2. Glycine max expressed sequence tags (ESTs) with homology to genes involved in the endogenous time-keeping mechanism of Arabidopsis thaliana Arabidopsis thaliana Glycine max Protein motifs and References biological process Name Gene EST % a e value CCA1 AT2G46830 AI (P: 48%) 2e-38 Negative limp, MYB DNA binding Wang et al domain ELF4 AT2G40080 CA (P: 42%) 1e Positive limb, plant specific Doyle et al DUF131 motif Kikis et al BG (P: 42%) 6e LHY AT1G01060 BE (P: 65%) 1e-40 Negative limb, MYB DNA Schaffer et al binding domain Alabadi et al LUX AT3G46640 AW (P: 60%) 6e-47 Putative component, MYB DNA binding Hazen et al domain PRR3 AT5G60100 BU (P: 28%) 8e-68 Rhythm regulator, response regulator Makino et al domain, CCT motif PRR5 AT5G24470 BM (P: 29%) 2e-59 Rhythm regulator, response regulator Nakamichi et al receiver domain, MYB DNA binding domain PRR7 AT5G02810 BU (P: 28%) 2e-69 Rhythm regulator, response regulator Nakamichi et al receiver domain, CCT motif Salomé and Mcclung 2005 PRR9 AT2G46790 BM (P:25%) 2e-53 Rhythm regulator, response regulator Salomé and Mcclung 2005 receiver domain TOC1 AT5G61380 TC (P: 15%) 1e Rhythm regulator, response regulator Strayer et al receiver domain, CCT motif TC (P: 28%) 4e Gene name abbreviations: CCA1, circadian clock-associated 1; ELF4, early flowering 4; LHY, long hypocotyl and early flowering 1; PRR, Arabidopsis pseudo-response regulator; TOC1, timing of CAB 1. Functional domains abbreviations: CCT, CONSTANS-like C-terminal; DUF, domain of unknown function. a Identity percentage at the amino acid level. seedlings and mature plants (Figure 3). The most frequent PRR transcripts in the soybean genome database were similar to PRR5/PRR9 and TOC1, although with distinct expression patterns (Figure 3). Arabidopsis ELF4 belongs to a family of several hypothetical plant proteins of around 100 residues in length with no functionally characterized domain. It is thought to be involved in an interlocking auto-regulatory transcriptional feedback loop that works in conjunction with, or parallel to, the CCA1, LHY and TOC1 loop (Kikis et al. 2005). The soybean genome has two EST contigs sharing sequence similarity with Arabidopsis ELF4 and with each other (95.3% deduced amino acid sequence identity). The clock negative limb MYB transcription factors CCA1 and LHY from Arabidopsis are represented in the soybean genome by two highly similar (48.9% deduced amino acid sequence identity) EST contigs (Table 2, Figure 4A,B). Soybean sequences are highly similar to the Arabidopsis MYB factors at the N-terminal binding site for the YAAC(G/T)G promoter element region (Figure 4A). Thus, GmCCA1 and GmLHY are likely to be functionally equivalent to their Arabidopsis counterparts. Moreover, these cdnas are highly represented in seedling libraries (Figure 5), which indicate that they may be involved in de-etiolation control and photomorphogenesis responses comparative to the one of AtCCA1 and AtLHY. Soybean homolog of Arabidopsis putative clock component MYB-factor LUX ARRYTHMO (LUX) is also highly represented in seedling-derived libraries (Table 2, Figure 5). Photoperiodic flowering induction pathway The photoperiod sensing transcription factor CO is a member of a 17 gene family in Arabidopsis, consisting of three clades referred to as groups I, II, and III (Griffiths et al. 2003). Group I genes (CO and CONSTANS-LIKE1 [COL1] COL5) have essentially same domain structure as CO, whereas group II genes (COL6 COL8 and COL16) have a single B-box, and group III genes (COL9 COL15) have the second B-box replaced by a more divergent zinc-finger domain (Griffiths et al. 2003). We have found nine soybean EST contigs showing intermediate to high levels of sequence similarity to the three groups of CO proteins (Figure 6). As expected, higher sequence

6 In Silico Candidates for Photoperiodism in Soybean 1645 Pseudo response regulator domain Figure 2. Pseudo response regulator family of circadian clock components in Glycine max. (A) Schematic representation of the domain structure of PRR proteins lacking the invariant phospho-accepting asparagine (replaced by a glutamate residue) and alignment of the pseudo response-regulator domain. (B) Phylogenetic analysis of PRR1/TOC1-like pseudo response regulator family. Dark gray and light gray boxes indicate sequence identity and similarity, respectively. Neighbor-joining trees for the deduced amino acid sequences from soybean aligned with ClustalX are shown. Bootstrap values are indicated above each branch. At, Arabidopsis thaliana. homology was found at the family consensus N-terminal bzip domains and at the CCT domain at the carboxy terminus (Figure 6B). In soybean, group I of CO presented a subdivision due to sequence divergence at the Zn-finger B-box domain (Figure 6A). Similar separation of the DNA binding domains of group I CO genes has been observed in previous studies with legume species (Hecht et al. 2005). The G. max CO family group II consisted of a higher number of EST contigs, however, higher sequence divergence was present at the single B-box domain and sub-grouping was observed even in the bootstrapped tree (Figure 6A). Group III was absent from the soybean CO family, possibly due to the highly stringent search criteria used. Sequences with lower similarity at the B-box and CCT domains were observed in soybean databases; however, they failed to retrieve the original Arabidopsis gene and were excluded from analyses (data not shown). The soybean genome has two EST contigs similar to the transcription factor CDF1 (CYCLING DOF FACTOR 1)

7 1646 Journal of Integrative Plant Biology Vol. 49 No flowering phenotype: ELF5, ELF6, ELF7 and ELF8 (EARLY FLOWERING). Similar to the Arabidopsis genes, soybean ELF sequences are unrelated to each other. Soybean ELF5 homolog has the two signature tryptophan (W) residues that are spaced amino acids apart as well as the conserved proline residue that constitute the signature WW domains. ELF6 is a Jumonji (Jmj)/Zn finger transcription factor in Arabidopsis and in soybean (Table 3). ELF7 and ELF8 show sequence similarities to the yeast five-member PAF1 complex that associates with RNA polymerase II and are thought to regulate gene expression by recruiting SET1 (a histone 3 Lys 4 [H3- K4] methyl transferase) to the initially transcribed 5 regions of target chromatin. In soybean, ELF7 and ELF8 are represented by two and one EST contigs, respectively. Soybean cdnas share more than 70% deduced amino acid sequence identity with their Arabidopsis homologs (Table 3). Figure 3. In silico expression profile of the PRR-like transcripts in soybean. The normalized number of reads for the transcripts in grouped libraries is represented as grayscale. Hierarchical clustering of the expression patterns is represented by the tree. (Table 3). The deduced amino acid sequences are closely related to each other and to the Arabidopsis paralog, especially at the Zn finger DNA-binding domain. The Dof domain has a significant resemblance to cysteine 2/ cysteine 2 Zn finger DNAbinding domains of steroid hormone receptors and GATA1, but has a longer putative loop where an extra cysteine residue is conserved. The deduced amino acid sequence of the soybean CDF1 cdnas shows fully conserved cys2/cys2 in the Zn finger domain (data not shown). The direct regulatory target of CO, the flowering promoter FLOWERING LOCUS T (FT), is also represented in the soybean genome (Table 3). The soybean homolog also shares weak sequence similarities with phosphatidylethanolamine binding protein or Raf-kinase inhibitor protein throughout the sequence. Similarly, we have identified in the soybean genome a homolog of the target of FT, the bzip transcriptional regulator FLOWERING LOCUS D (FD) (Table 3), although the potential phosphorylation site for Ca 2+ -dependent protein kinases (CDPKs) the C terminus of FD (RSST, 279 to 282aa) and associated with its function is absent from the soybean homolog. The floral pathway integrator SUPPRESSOROFCONSTANS 1 (SOC1) is represented in soybean by four highly similar EST contigs (Table 3) whereas in Arabidopsis the clade containing SOC1 consists of five other members. Soybean sequences are highly similar to AtSOC1 at the amino-terminus, which contains the K-box region, SRF-type DNA binding domain and the signature bzip region. We have identified soybean homologs of several flowering repressors from Arabidopsis whose mutants present an early Discussion Input pathway genes In soybean, the identified photoreceptor gene families are likely to be involved in the input pathway to the clock due to their extensive sequence similarity to the Arabidopsis homologs (Quecini et al. 2007). Moreover, classic genetic and physiological studies have demonstrated the influence of light quality in photoperiodic flowering induction (Cober et al. 1996; Cober and Voldeng 2001). Candidate gene approaches have suggested a link between soybean maturity locus E1 and phyb (Cober et al. 1996), although their map position is distinct (Tasma and Shoemaker 2003). In Arabidopsis, light quality control of flowering time is dependent of the nuclear protein PFT1 that functions in a phybdependent manner (Cerdán and Chory 2003). Interestingly, soybean has a cdna with deduced amino acid sequence highly homologous to PFT1 (more than 80% identity). Shadeavoidance responses, also triggered by low R:FR rates, are independent of PFT1 in Arabidopsis (Cerdán and Chory 2003). Correspondingly, soybean E alleles do not cause early flowering under shade-avoidance inducing conditions (Cober and Volgende 2001). Thus, the observed discrepancy between the map position of soybean maturity locus E1 and candidate gene PHYB may be explained by the presence of a PFT1-based light quality pathway modulating soybean photoperiodic flowering. Arabidopsis ELF3 physically interacts with phyb and controls the gating mechanism to the clock input (Covington et al. 2001; Hicks et al. 2001; Liu et al. 2001). In soybean, ELF3 is represented by three EST contigs with intermediate similarity tothearabidopsis ortholog. The absence of functionally characterized protein domains in the protein prevents further analysis of GmELF3 genes, although the acidic proline, threonine and glutamine-rich regions characteristic of Arabidopsis ELF3 are present in the soybean homolog. At this point, the existence

8 In Silico Candidates for Photoperiodism in Soybean 1647 Figure 4. MYB-type transcription factors involved in the circadian clock mechanism in Glycine max. (A) Schematic representation of the domain structure of MYB depicting the conserved SHAQK motif and alignment of the MYB DNA-binding domain. (B) Phylogenetic analysis of CCA1/LHY-like family. Dark gray and light gray boxes indicate sequence identity and similarity, respectively. Neighborjoining trees for the deduced amino acid sequences from soybean aligned with ClustalX are shown. Bootstrap values are indicated above each branch. At, Arabidopsis thaliana. of soybean ELF3 orthologs with higher degrees of sequence similarity cannot be ruled out. Alternatively, distinct proteins may be responsible for the gating mechanism to the soybean circadian clock. Central oscillator feedback loop genes The oscillatory mechanism of circadian clocks consists of autoregulatory transcriptional feedback loops, with positive factors activating the transcription of negative factors, which will in turn repress the transcription of positively acting factors until their level decreases and the cycle restarts (Bell-Pedersen et al. 2005). Arabidopsis central oscillator bona fide components are the pseudo-response regulator TOC1/PRR1 (Makino et al. 2000; Strayer et al. 2000) in the positive limb, and the plantspecific protein ELF4 (Kikis et al. 2005) and the MYB-domain transcription factors LHY and CCA1 (Schaffer et al. 1998; Wang and Tobin 1998) in the negative limb. During the late evening, TOC1 activates the expression of CCA1/LHY (Alabadí et al. 2001), higher levels of CCA1/LHY allow them to bind to the evening element (EE, AAATATCT) at the promoter of TOC1 (Harmer et al. 2000) and repress its transcription, decreasing the levels of CCA1 and LHY during the day. In turn, lower levels of CCA1 and LHY release the inhibition on the expression of TOC1, which peaks at dusk when CCA1 and LHY levels are at their trough (Alabadí et al. 2001). During the evening, TOC1 and the MYB-domain factor LUX ARRYTHMO accumulate and promote the expression of CCA1 and LHY (Alabadí et al. 2001; Hazen et al. 2005), closing the circle. Recently, an ancillary interlocked loop consisting of ELF4 and CCA1/LHY has been

9 1648 Journal of Integrative Plant Biology Vol. 49 No Figure 5. In silico expression profile of the Glycine max transcripts with homology to MYB transcription factors implicated in the central oscillator mechanism of the endogenous clock in Arabidopsis thaliana. The normalized number of reads for the transcripts in each library is represented as grayscale. Hierarchical clustering of the expression patterns is represented by the tree. proposed (Kikis et al. 2005). The identification of soybean homologs for Arabidopsis bona fide oscillator components (Table 2) indicates a functional conservation in the endogenous time-keeping system between these species. In Arabidopsis, TOC1/PRR1 is a member of the six-gene PRR family, whose mrna abundance oscillates with circadian periodicity for all the members, except for PRR2 (Matsushika et al. 2000; Strayer et al. 2000). Interestingly, the soybean PRR family appears to have undergone a differential gene expansion process during evolution, since it consists of three members: one closer to PRR3 and PRR7, one more similar to PRR5 and PRR9 and a third one similar to PRR1/TOC1. In Arabidopsis, PRR5 and PRR9 display extensive sequence similarity, however, studies with loss-of-mutants have demonstrated the lack of functional overlap (Erickson et al. 2003). Similarly, PRR7 and PRR9, which have more divergent sequences, have been demonstrated to be partially redundant roles in the Arabidopsis circadian clock (Salomé and McClung 2005). The divergent expression pattern of soybean PRR-like transcripts suggests that they might be carrying out distinct functions in agreement with the phylogenetic analyses. However, at this point the existence of other PRR family genes in the soybean genome cannot be ruled out. The transcriptional regulation within the Arabidopsis oscillatory clock mechanism relies on MYB-type transcription factors, namely CCA1, LHY, and LUX. These proteins are members of a unique subfamily of putative MYB transcription factors with a single MYB-DNA binding domain. The single MYB domains found in CCA1, LHY, and LUX are classified as SHAQKYFtype; however, these three proteins do not constitute a gene family due to the distinct structure, sequence and expression regulation of LUX (Hazen et al. 2005). In soybean, we have identified sequences highly homologous to the Arabidopsis MYB factors associated with the circadian clock function. The newly identified component of the oscillatory mechanism of Arabidopsis circadian clock, ELF4, is a member of a plantspecific protein family of 111 kda with no recognizable domain (Doyle et al. 2002). It functions in an interlocked feedback loop activating, in an ELF3-dependent manner, the transcription of CCA1 and LHY, which in turn, represses ELF4 transcription (Kikis et al. 2005). Soybean has two highly similar ELF4 orthologs (more than 95% deduced amino acid sequence identity), however the sequence conservation to the Arabidopsis functionally characterized protein is lower. The absence of functionally recognizable domains in AtELF4 prevents the establishment of further valid comparisons between the model system and soybean without functional analyses. Moreover, the soybean genome has not been fully sequenced, which makes it likely that small transcripts with low expression levels such as ELF4 are still absent from current databases. Photoperiodic flowering induction pathway Soybean is a vernalization-unresponsive SD species (Summerfield and Roberts 1985) and classic genetic studies have demonstrated that the transition to the reproductive stage is controlled by separated genetic pathways determining the length of the juvenile period and the photoperiodic induction (Destro et al. 2001). The control of CO protein expression and activity monitors photoperiodic changes in Arabidopsis (Yanovsky and Kay 2002; Valverde et al. 2004) and in rice (Hayama and Coupland 2004). High levels of CO mrna coincide with light under long days, leading to an increase in FT expression (Suárez-López et al. 2001). The photoperiodic response rhythm is provided by the rhythm of CO expression, and light input to FT is perceived by PHYA and CRY2 (Yanovsky and Kay 2002). The rhythmic expression of CO is dependent on the degradation of its repressor CDF1 by the F-box protein FKF1 (Imaizumi et al. 2003; Imaizumi et al. 2005). In soybean, a similar mechanism is possible due to the presence of CO, CDF1, FKF1 and FT homologs.

10 In Silico Candidates for Photoperiodism in Soybean 1649 Figure 6. CONSTANS/CONSTANS-LIKE family in Glycine max. (A) Phylogenetic analysis and molecular structure. (B) Sequence alignment of CO/COL gene family showing DNA binding B-box and CCT terminal domain. Neighbor-joining trees for a concatenation of the Zn finger B-box domains and CCT domains (207 characters) aligned with ClustalX are shown. Bootstrap values are indicated above each branch. At, Arabidopsis thaliana. Soybean FKF1- and CO-like transcripts are highly similar to their Arabidopsis orthologs. Interestingly, a previous survey of soybean sequences has been unable to identify FT orthologs in soybean (Hecht et al. 2005) due to the relatively low level of sequence identity. In Arabidopsis, CO suppressor CDF1 is a member of a three-gene family with circadian-regulated expression profile (Imaizumi et al. 2005). Two highly similar CDF1- like sequences were found in soybean, suggesting a functional conservation with their Arabidopsis counterparts. In Arabidopsis, the CO/COL family consists of three clades: one with a single Zn finger DNA binding B-box and two with two B-box domains (B1 and B2). In some proteins, B2 sub-domain is highly divergent allowing the separation of the group into two clades. Soybean CO/COL bona fide orthologs were grouped to clades I and II. Interestingly, one soybean transcript (BQ785772) has a mildly divergent B2 domain and may represent an intermediate between conserved B-boxes in clade I and more divergent ones in clade III that is absent in Arabidopsis. The identification of components from the flowering enabling (FLC and SVP) and photoperiodic-inductive (CO, SOC1 and FT) pathway in soybean may provide mechanistic insights into the function of the known flowering-time G. max alleles. The floral repressors ELF5, ELF6, ELF7 and ELF8 are also represented in the soybean genome. In Arabidopsis, ELF5 functions to regulate FLC levels, possibly by an RNA-binding interaction, thus, affecting the autonomous and photoperiodic flowering

11 1650 Journal of Integrative Plant Biology Vol. 49 No Table 3. Glycine max expressed sequence tags (ESTs) with homology to genes acting in the photoperiodic flowering induction from Arabidopsis thaliana. Arabidopsis thaliana Glycine max Protein motifs and Reference biological process Name Gene EST % e value CDF1 AT5G62430 BM (P: 34%) 1e Dof Zn finger transcriptional regulator Imaizumi et al BU (P: 29%) 4e CO AT5G15840 BE (P: 49%) 2e-35 Zn finger B-box DNA binding motif, putative Putterill et al. 1995; Zinc finger C2HC5, CCT motif Griffiths et al ELF5 AT5G62640 BM (P: 26%) 8e-53 WW domain (Npw)38-binding protein Noh Y-S. et al (NpwBP), photoperiodic flowering repressor ELF6 AT5G04240 CX (P: 37%) 1e-15 Jmj /Zn finger transcriptional regulator, Noh B. et al photoperiod pathway negative regulator ELF7 AT1G79730 AW (P: 25%) 1e PAF1 homolog, recruits a histone He et al AW methyl transferase, FLC regulator 72.8 (P: 37%) 4e ELF8 AT2G06210 AW (P: 35%) 4e-38 CTR9 homolog, recruits a histone methyl He et al transferase, FLC regulator FD AT4G35900 BE (P: 31%) 1e-15 bzip transcriptional regulator, Abe et al. 2003; Halliday 2003 spatial-temporal integration photoperiodic flowering stimulus FLC AT5G10140 AI (P: 72%) 1e-33 MADS box transcriptional regulator Michaels and Amasino 2000 FT AT1G65480 CX (FL) 5e-58 Phosphatidylethanolamine-binding Izawa et al. 2002; protein motif Wigge et al SOC1 AT2G45660 BE (P: 95%) 5e K-box region, SRF-type transcription factor, Onouchi et al bzip DNA binding motif, plant specific DUF1319 motif BE (P: 65%) 2e AW (P: 64%) 6e AW (P: 88%) 2e SVP AT2G22540 AY (P: 60%) 1e-31 SRF-type transcription factor, K-box motif Hartman et al Gene name abbreviations: CDF1, cycling Dof factor 1; CO, CONSTANS; ELF6, early flowering 6; FT, flowering locus T; PIL, PIF-like; SOC1, suppressor of CONSTANS 1. Functional domains abbreviations: bhlh, basic helix-loop-helix; bzip, basic leucine zipper; CCT, CONSTANS-like C-terminal; DUF, domain of unknown function; E-MAP-115, epithelial cells tissue specific microtubule-associated protein of 115 kda; SRF, human serum responsive factor: WW domain, conserved tryptophan; Zn finger, zinc finger. Identity percentage at the amino acid level. pathways (Noh Y-S et al. 2004). ELF6 is Jumonji/Zn finger transcriptional regulator and acts as an upstream repressor in the photoperiodic flowering pathway (Noh B et al. 2004). The observed interactions between the length of the juvenile period and the photoperiodic response would accommodate roles for ELF5 and ELF6 in soybean flowering pathways. ELF7 encodes the Arabidopsis homolog of PAF1, and ELF8 encodes the relative of yeast CTR9, members of a complex of five proteins that associate with RNA Pol II in yeast (Mueller et al. 2004). In Arabidopsis, ELF7 and ELF8 are required for histone methylation of FLC and of its homolog FLM, thus inducing their expression and repressing flowering (He et al. 2004). The unresponsive vernalization characteristic of soybean prevents us to promptly attribute function to ELF7 and ELF8 ortologs. In Arabidopsis, photoreceptors are considered to mediate the thermosensory pathway controlling flowering (Blázquez et al. 2003), whereas in soybean, ambient temperature has more effect to influence flowering at night (Garner and Allard 1933; Parker and Borthwick 1950), thus indicating that this response is independent of photoreceptors in this species. Thus, ELF7 and ELF8 homologs may be candidates for this function in soybean, since they function downstream of the photoperiodic pathway. Concluding remarks This preliminary survey of soybean genes involved in the endogenous time-keeping mechanism and in the photoperiodic

12 In Silico Candidates for Photoperiodism in Soybean 1651 control of flowering time has provided useful information for further studies. Moreover, it has allowed the identification of a genetic framework controlling the transition from vegetative to reproductive growth. These findings are crucial to advance soybean breeding programs concerned with the photoperiodic flowering induction, which determines the crop latitudinal and seasonal establishment. An immediate goal of plant genomics is to transfer knowledge between model and crop species, contributing to the understanding of the mechanisms underlying several aspects of plant physiology. Thus, this study will help to elucidate how environmental factors modulate plant development and the expression of phenotypic characters. Our results give a new perspective on several aspects of photoperiodic flowering induction in soybean. Material and Methods Database searches and alignments Homologs of Arabidopsis thaliana circadian clock and photoperiodic flowering control genes were identified in BLAST searches (Altschult et al. 1997) against soybean and legume index databases at NCBI (National Center for Biotechnology Information TIGR (The Institute for Genomic Research Soybean Genomics Initiative at the University of Minnesota ( at Iowa State University ( at Washington University in St. Louis ( and at USDA/ARS Soybase ( Data validation was carried out by tblastx and tblastn searches of the soybean sequence against the Arabidopsis genome database (The Arabidopsis Information Resource; The resulting alignments were filtered by a threshold e-value of 1e-15 for the hits and further analyzed according to functional domain description. Validated sequences were translated and protein (deduced amino acid) alignments were carried out using ClustalX (Thompson et al. 1997). When necessary, alignments were manually adjusted using Lasergene MegAlign (DNASTAR, Madison, WI, USA, Motif analysis and in silico characterization The identified homologs were further investigated for the presence and sequence conservation of recognizable functional domains described in several protein analysis databases (European Bioinformatics Institute-European Molecular Biology Laboratory EMBL-EBI Expert Protein Analysis System ExPaSy do Swiss Institute of Bioinformatics SIB and Protein Families database Pfam Phylogenetic analysis The putative functionality of the G. max genes in comparison to their Arabidopsis homologs was assessed by genetic distance and phylogenetic studies. Phylogenetic analyses were carried out using distance and parsimony methods in the software PAUP 4.0b10 ( using the software default parameters. Re-sampling bootstrap trees containing random samples were constructed using PSIGNFIT software ( Modular functional domains were used for genetic distance studies for genes previously characterized as having divergent regions and conserved blocks. In silico gene expression analysis Qualitative gene expression profiling was carried out by in silico analyses of the aforementioned soybean EST databases using virtual northern blot (VNB) analyses. VNB used BLAST to query the input gene against other sequences in reference sequence databases, generating an alignment of the input gene to its paralogs. The resulting alignment is then used to find sequences in the entire mrna input that are specific to the input gene (probes), which are used collectively, to query the EST databases again using BLAST to avoid false-positives, or ESTs which come from a paralog of the input gene rather than the input gene itself. The identity numbers of the ESTs matching the probes are recovered and NCBI ( is used to find the names of the libraries from which those ESTs were derived and to identify a tissue for each library. References Abe J, Xu D, Miyano A, Komatsu K, Kanazawa A, Shimamoto Y (2003). Photoperiod-insensitive Japanese soybean landraces differ at two maturity loci. Crop Sci. 43, Alabadí D, Oyama T, Yanovsky MJ, Harmon FG, Más P, Kay SA (2001). Reciprocal regulation between TOC1 and LHY/CCA1 within the Arabidopsis circadian clock. Science 293, Alabadí D, Yanovsky MJ, Más P, Harmer SL, Kay SA (2002). Critical role for CCA1 and LHY in maintaining circadian rhythmicity in Arabidopsis. Curr. Biol. 12, Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W et al. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucl. Acids Res. 25, Bell-Pedersen D, Cassone VM, Earnest DJ, Golden SS, Hardin PE, Thomas TL et al. (2005). Circadian rhythms from multiple oscillators: lessons from diverse organisms. Nat. Rev. Genet. 6, Bernard RL (1971). Two genes for time of flowering and maturity in soybeans. Crop Sci. 11, Blázquez M, Ahn JH, Weigel D (2003). A thermosensory pathway controlling flowering time in Arabidopsis thaliana. Nat. Genet. 33,

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