Biosci. Biotechnol. Biochem., 77 (4), 747–753, 2013 Clock-Controlled and FLOWERING LOCUS T (FT)-Dependent Photoperiodic Pathway in Lotus japonicus I: Verification of the Flowering-Associated Function of an FT Homolog y Takafumi YAMASHINO, Saori YAMAWAKI, Emi HAGUI, Hanayo UEOKA-NAKANISHI, Norihito NAKAMICHI, Shogo ITO, and Takeshi MIZUNO Laboratory of Molecular and Functional Genomics, School of Agriculture, Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan Received November 13, 2012; Accepted January 14, 2013; Online Publication, April 7, 2013 [doi:10.1271/bbb.120871] During the last decade, significant research progress highly conserved throughout the plant kingdom.5–8) in the study of Arabidopsis thaliana has been made in However, it is also assumed that the mechanisms by defining the molecular mechanism by which the plant which the clock regulates flowering time differs consid- circadian clock regulates flowering time in response to erably in detail between species.9–11) In this respect, changes in photoperiod. It is generally accepted that comparative genomics approach might be effective in the clock-controlled CONSTANS (CO)-FLOWERING addressing these issues. LOCUS T (FT)-mediated external coincidence mecha- A. thaliana is classified as a facultative annual long- nism underlying the photoperiodic control of flowering day plant, whose flowering is induced in response to a time is conserved in higher plants, including A. thaliana longer day length (or photoperiod). The circadian clock and Oryza sativa. However, it is also assumed that the can measure the day length, and tells the time to mechanism differs considerably in detail among species. flower.3,4,12) The key player in the photoperiodic Here we characterized the clock-controlled CO-FT induction of flowering is CONSTANS (CO), which pathway in Lotus japonicus (a model legume) in serves as a DNA-binding transcription activator.13,14) comparison with that of A. thaliana. L. japonicus has The transcription of CO is under the control of the at least one FT orthologous gene (named LjFTa), which circadian clock and shows a biphasic diurnal expression is induced specifically in long-days and complements the profile with peaks in late daytime and nighttime mutational lesion of the A. thaliana FT gene. However, specifically in LDs.15–17) It has been found that CO it was speculated that this legume might lack the protiens are stabilized in late daytime.18–20) Hence, CO upstream positive regulator CO. By employing L. japo- can actively promote the transcription of FLOWERING nicus phyB mutant plants, we showed that the photo- LOCUS T (FT) in leaf phloem only in LDs.12) FT receptor mutant displays a phenotype of early flowering gene-products act in the shoot apical meristem to due to enhanced expression of LjFTa, suggesting that induce reproductive growth.21) In SDs, however, CO LjFTa is invovled in the promotion of flowering in is transcribed excluisively in nighttime. Since the CO L. japonicus. These results are discussed in the context gene-products are degrated by CONSTITUTIVE of current knowledge of the flowering in crop legumes PHOTOMORPHOGENIC 1 (COP1) in nighteime, FT such as soybean and garden pea. is not transcribed in short-days (SDs).19) In short, the clock-contolled CO-FT pathway is essential in promot- Key words: Arabidopsis thaliana; circadian clock; flow- ing flowering predominantely in an appropriate season ering time; Lotus japonicus; photoreceptor (or LDs) in A. thaliana. mutant We have been studying circadian clock-controlled biological mechanisms, including photoperiodic control Since plants are sessile, they must be able to sense of flowering time, employing not only A. thaliana but changes in environmental light conditions and adapt also L. japonicus.22–25) L. japonicus belongs to a large their developmental processes accordingly. In this family of legumes that are present in most ecosystems respect, the circadian clock plays prominent roles and include many important crop species.26) It is a model through providing an adaptive advantage in anticipating legume of choice in conducting comparative genome- daily changes in light/dark conditions and seasonal wide studies,27–29) together with Medicago truncatula,30) changes in the photoperiod.1,2) In Arabidopsis thaliana, Glycine max (soybean),31,32) and Pisum sativum (garden significant progress has been made in defining the pea).33) Among these, Medicago and Pisum are predom- molecular mechanisms by which the circadian clock inantly long day plants from temperate regions, whereas regulates long-days (LDs)-specific promotion of flower- soybean as well as bean (Phaseolus) originating at lower ing in A. thaliana.3,4) It is generally accepted that the latitudes is predominantly short-days plants. L. japoni- molecular bases of clock-controlled flowering time are cus (accessions Gifu and Miyakojima MG-20) is a y To whom correspondence should be addressed. Fax: +81-52-789-4091; E-mail: [email protected] Abbreviations: CaMV, cauliflower mosaic virus; CO, CONSTANS; COP1, CONSTITUTIVE PHOTOMORPHOGENIC 1; FLC, FLOWERING LOCUS C; FT, FLOWERING LOCUS T; LDs, long-days; SDs, short-days 748 T. YAMASHINO et al. perennial temperate legume that sets flowers preferen- The BLAST conditions used were blastp against all predicted peptides, tially in LDs.34,35) It is important to understand how the filter low complexity, expect value 1, and matrix BLOSUM62. The circadian clock regulates the flowering time of legume best-hit coding sequence in L. japonicus was found, and the results were summarized in Supplemental Table S1 (see Biosci. Biotechnol. species in repose to seasonal changes in the photoperiod, Biochem. Web site). because this has a strong impact on the yields of crops. It would also be important to understand how the Plant materials and growth conditions. A. thaliana Columbia-0 and mechanisms underlying the regulation of flowering time L. japonicus Miyakojima MG-20 were used in this study. The were modified optionally in L. japonicus during evolu- L. japonicus experimental strain, Miyakojima MG-20, was provided tion to adapt to domestic habitates. by the National BioResource Project (L. japonicus and G. max). The L. japonicus phyB Among legumes, pea and soybean have long been mutant (EMS mutagenesis line 01-0017) was provided by Dr. A. Suzuki (Saga University). All analyses were characterized in terms of the control of flowering time, conducted in a cultivation chamber (MLR-350, Panasonic or LPH-350, and recently, a small family of FT homologs in legumes Nippon Medical & Chemical Instruments) under neutral white has been characterized for M. truncatula, pea, and fluorescent light at a constant temperature of 22 C and 25 C for soybean, based on genome-wide information.33,36–40) A. thaliana and L. japonicus respectively. The intensity of the light These FT homologs of legumes are commonly classified was adjusted to 80 mmolÁm2ÁsÀ1 for A. thaliana, and to more than 2 À1 into three clades, designated FTa, FTb, and FTc, 180 mmolÁm Ás for L. japonicus. A daily 16 h light/8 h dark cycle was adopted as the long day condition of A. thaliana and L. japonicus. according to phylogenetic analyses. Some of them have Daily 10 h light/14 h dark and 11.5 h light/12.5 h dark cycles were been demonstrated that they have an ability to promote adopted as the short day conditions of A. thaliana and L. japonicus 37) flowering. We also previously showed that a gene respectively. For flowering assay, plants were germinated and grown encoding a putative FT homolog in L. japonicus has an on a gellan gum plate containing MS salts and 1% sucrose for a week, ability to promote flowering in A. thaliana.24) Hence it and then seedlings were transferred to soil. is conceivable that the function of FT homologs is Preparation of RNA and qRT-PCR. conserved between A. thaliana and legumes, but it is Total RNA was purified from frost plant (100 mg) with an RNeasy plant mini kit (Qiagen). To speculated that there might be significant differences in synthesize cDNA, RNA (1 mg of each) was converted to cDNA with detail of the mechanisms underlying the regulation of ReverTra Ace (TOYOBO) and oligo-dT primer. The synthesized flowering time between A. thalana and legumes. It is cDNAs were amplified with SYBR Premix Ex Taq II (Takara Bio) well-known that FLOWERING LOCUS C (FLC), which and the primer set for each target gene, analyzed by using a Stepone is involved in repression of FT in the vernalization PlusÔ Real-Time PCR System (Life technologies). The primer pathway of flowering, belongs to a specialized MIKC- sets used were described in Supplemental Table S2. LjUBC (chr1.LjT04O06.110.r2.m), encoding an ubiquitin carrier protein, was type MADS box transcriptional factor that evolved only used as internal references. The following standard thermal cycling in Brassica species. Moreover, the result of an extensive program was used for all PCR: 95 C for 120 s, 40 cycles of 95 C for inspection of the soybean, L. japonicus, and M. trunca- 10 s, and 60 C for 60 s. The CT values for individual reactions were tula genome seequences suggests that these legumes determined by analysis of the raw fluorescence data (without baseline seem to lack the orthologous gene CO.24,37) Considering correction) using the freely available software PCR Miner (http: 41) these, it is important to clarify whether the canonical //www.miner.ewindup.info). Based on the comparative CT method, CO-FT-mediated flowering pathway is conserved in relative expression level was calculated. L. japonicus. Phylogenetic analysis. Amino acid sequences deduced from the For this purpose, we previously compiled a list of determined cDNA sequences were aligned using the ClustalW genes presumably implicated in the FT-mediated photo- program, and the numbers of amino acid substitutions between each periodic control of flowering time in L.