Functional Analysis of FT and TFL1 Orthologs from Orchid ( Oncidium Gower Ramsey) that Regulate the Vegetative to Reproductive Transition

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1 Regular Paper Functional Analysis of FT and TFL1 Orthologs from Orchid ( Oncidium Gower Ramsey) that Regulate the Vegetative to Reproductive Transition Cheng-Jing Hou and Chang-Hsien Yang Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan 7 ROC The FLOWERING LOCUS T (FT) and TERMINAL FLOWER 1 ( TFL1 ) genes play crucial roles in regulating the vegetative to reproductive phase transition. Orthologs of FT/TFL1 ( OnFT and OnTFL1 ) were isolated and characterized from Oncidium Gower Ramsey. OnFT mrna was detected in axillary buds, leaves, pseudobulb and flowers. In flowers, OnFT was expressed more in young flower buds than in mature flowers and was predominantly expressed in sepals and petals. The expression of OnFT was regulated by photoperiod, with the highest expression from the 8th to 1th hour of the light period and the lowest expression at dawn. In contrast, the expression of OnTFL1 was only detected in axillary bud and pseudobulb, and was not influenced by light. Ectopic expression of OnFT in transgenic Arabidopsis plants showed novel phenotypes by flowering early and losing inflorescence indeterminacy. In addition, ectopic expression of OnFT was able to partially complement the late flowering defect in transgenic Arabidopsis ft-1 mutants. In transgenic tfl1-11 mutant plants, 3S:: OnTFL1 delayed flowering and rescued the phenotype of terminal flowers. Furthermore, substitution of the key single amino acid His8 by Tyr was able to convert the OnTFL1 function to OnFT by promoting flowering in 3S:: OnTFL1-H8Y transgenic Arabidopsis plants. Further analysis indicated that the expression of APETALA1 (AP1) was significantly up-regulated in 3S:: OnFT and 3S:: OnTFL1-H8Y plants, and was downregulated in 3S:: OnTFL1 transgenic Arabidopsis plants. Our data indicated that OnFT and OnTFL1 are putative phosphatidylethanolamine-binding protein genes in orchids that regulate flower transition similar to their orthologs in Arabidopsis. Keywords: Arabidopsis thaliana FLOWERING LOCUS T (FT) flower transition Oncidium Gower Ramsey TERMINAL FLOWER 1 (TFL1) Abbreviations : CaMV, cauliflower mosaic virus ; RACE, rapid amplified cdna ends. Introduction The initiation of flowering in higher plants is mainly controlled by four major pathways, including photoperiod, vernalization, hormone and autonomous regulation (for reviews, see Levy and Dean 1998, Simpson et al ). These multiple pathways converge on a small set of flowering timing genes and are influenced by environmental signals. Of these genes, FLOWERING LOCUS T (FT ), a well-known floral integrator gene, plays an important role in controlling flowering time ( Kardailsky et al. 1999, Kobayashi et al. 1999, Samach and Wigge, for a review, see Komeda ). FT encodes a protein with high similarity to RAF kinase inhibitor proteins (RKIP) ( Kardailsky et al. 1999, Kobayashi et al ) and is in the phosphatidylethanolamine-binding protein (PEBP) gene family, which includes TERMINAL FLOWER 1 (TFL1 ), BROTHER OF FT AND TFL1 ( BFT ), TWIN SISTER OF FT (TSF ), MOTHER OF FT AND TFL1 (MFT ) and ARABIDOPSIS THALIANA CENTRORADIALIS (ATC ) (Bradley et al. 1997, Ohshima et al. 1997, Kobayashi et al. 1999, Yoo et al., Yamaguchi et al., Mimida et al. 9 ). FT is partially functionally redundant with TSF to promote flowering and is regulated by CONSTANS (CO ), which encodes two B-box zinc-finger nuclear proteins and plays a central role in the integration of temperature and light in flowering Corresponding author: , chyang@dragon.nchu.edu.tw ; Fax, ; Plant Cell Physiol. (8): 1 17 (9) doi:1.193/pcp/pcp99, available online at The Author 9. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please journals.permissions@oxfordjournals.org 1

2 FT and TFL1 orthologs from Orchid control ( Putterill et al. 199, Huq et al., Onouchi et al., Suárez-López et al. 1, Halliday et al. 3, Valverde et al., Michaels et al. ). Expression of CO mrna leads to the induction of FT expression in phloem companion cells ( An et al. ), particularly in the distal minor veins of source leaves ( Takada and Goto 3 ), and causes a dramatic effect on the promotion of flowering ( Samach et al. ). Ectopic expression of either CO or FT has been shown to cause an early flowering phenotype in transgenic plants ( Kardailsky et al ). Thus, flowering regulation is established by a photoperiodic pathway through the transcriptional expression of GI-CO-FT ( Fujiwara et al. 8 ). More recent reports have implied that FT protein is a movable signal transducer known as florigen. FT and variant orthologs such as proteins Hd3a of rice and Cm-FT of cucurbits are able to move long distances through phloem to shoot apexes ( Corbesier et al. 7, Jaeger and Wigge 7, Lin et al. 7, Tamaki et al. 7 ) and interact directly with the bzip transcription factor FLOWERING LOCUS D (FD ) in shoot apex meristem (SAM) ( Abe et al. ). It has been demonstrated that FT interacts with FD to form a FT/FD heterodimer that binds to the promoter of APETALA1 (AP1 ) to activate flowering initiation ( Abe et al., Wigge et al. ). In addition to controlling flowering time, it has been shown that PtFT1, an aspen tree FT ortholog from Populus trichocarpa, played a novel role in the regulation of the short-day (SD)-induced growth cessation and bud set occurring in the fall ( Böhlenius et al. 6 ). In P. trichocarpa, flowering is induced by long days and is correlated with high PtFT expression in the spring and early summer, whereas growth cessation and bud set are induced in the fall and correlated with down-regulation of PtFT expression ( Böhlenius et al. 6 ). TERMINAL FLOWER 1 ( TFL1 ) also encodes a protein similar to RKIP ( Bradley et al ). However, the function of TFL1 is to maintain the inflorescence architecture by acting as a repressor for floral initiation and maintaining the inflorescence meristem through suppression of the expression of AP1 and LEAFY (LFY ) ( Bradley et al. 1997, Ohshima et al. 1997, Nilsson et al. 1998, Boss et al. ). Mutation in TFL1 produced terminal flowers and early flowering similar to that observed in 3S:: FT plants ( Bradley et al. 1997, Kardailsky et al ). TFL1 mrna is detected in and restricted to the inner cells of the SAM. However, a current model has shown that TFL1 proteins move to lateral regions, interact with FD and suppress AP1 and LFY to inhibit floral initiation and the conversion of the inflorescence meristem into a terminal flower ( Ahn et al. 6, Conti and Bradley 7 ). Thus, FT and TFL1 are mobile signals with opposite functions and are able to interact with FD to control floral meristem and inflorescence shoot development. Oncidium Gower Ramsey is an important economic orchid plant in flower markets around the world, including both potted and cut flowers. Little research has been reported on the role of MADS box genes in regulating flower transition and flower initiation in orchids ( Hsu and Yang, Hsu et al. 3 ). There have been no reports investigating the functions of flowering time genes of orchids. We report here the isolation and functional analysis of orthologs for FT and TFL1 genes from O. Gower Ramsey. The ectopic expression of these two genes that either promote or delay flowering in Arabidopsis are demonstrated. Furthermore, we provide evidence that the alteration of flowering time by these two genes in transgenic Arabidopsis plants is due to the induction or suppression of the flower meristem identity gene AP1. Results Isolation of OnFT and OnTFL1 cdna from O. Gower Ramsey Arabidopsis FT and TFL1 belong to the PEBP/RKIP family. Degenerate primers based on the conserved regions of the PEBP family were designed. A combined RT PCR and RACE strategy was used to clone OnFT and OnTFL1 from O. Gower Ramsey. OnFT cdna encodes a 176 amino acid protein that shows 7 %, 79 % and 8 % identity to Arabidopsis FT, rice Hd3a and Populus nigra PnFT ( Igasaki et al. 8 ), respectively (Fig. 1A ). The conserved key amino acid residue Tyr in FT homologs was indentified at position 8 of the OnFT protein ( Fig. 1A ). The cdna sequence of OnTFL1 encodes a 173 amino acid protein that shows 71 %, 8 % and 8 % identity to Arabidopsis TFL1, rice OsCEN and Zea mays ZmTFL1 ( Danilevskaya et al. 8 ), respectively ( Fig. 1A ). Similar to the conserved residue of the TFL1 homologs ( Hanzawa et al. ), His was identified at position 8 in OnTFL1 ( Fig. 1A ). Based on the protein sequence, OnFT and OnTFL1 share 9 % identity to each other ( Fig. 1A ). The sequence similarity between OnFT/OnTFL1 and FT/TFL1 indicates that OnFT/ OnTFL1 are the putative O. Gower Ramsey FT/TFL1 orthologs. The amino acid sequence alignment shown in Fig. 1A and the sequences for several other FT/TFL1 orthologs were used to construct a phylogenetic tree for the FT/TFL1 group genes ( Fig. 1B ). OnFT was assigned within the monocotyledonous subgroup of FT and is closely related to Hd3a of rice. OnTFL1 was assigned to the monocotyledonous subgroup of TFL1 and closely resembles OsCEN of rice. Gene expression of OnFT and OnTFL1 To further explore the relationship between sequence similarity and gene expression patterns, the detection of OnFT and OnTFL1 expression is necessary. As shown in Fig. 3A, OnFT mrna was detected in axillary buds ( Fig. A ) and leaves ( Fig. B ) during the vegetative stage. Similarly, OnFT mrna was detected in pseudobulb and mature leaves ( Fig. C ) during the reproductive stage ( Fig. 3A ). The expression level 1

3 C.-J. Hou and C.-H. Yang A OnFT MN----RERDSLIVGRVIGDVLDPFTRSVSLRVTYT-TRCITNGLELKPSVVVEQPRVEVGGNDLRTFYTLVMVDPDAPSPSNPQLREYLHWLVTDIPAT Hd3a.AG-SG.D..P.V...V...A.V..TN.K...G-SKTVS..C...M.TH...M...D.N...G. FT.S---INI..P...S..V...N..IT.K...G-Q.EV...D.R..Q.QNK...I..E...N...V...H... PnFT.S----.D..P.S...K.I...S-S.EVN..C...Q.AN...DI..E...D.S... OnTFL1.A----.AIEP.V...E..EL.NP..KMK...NCNKHVY..H..Y...ILDM...Q.G...S.F..I.T...V.G..D.Y...HV..I...G. OsCEN.S----.VLEP...K...E...N.NPT.KMTA..GANKQVF..H.FF..A.AGK...Q.G...S.F...T...V.G..D.Y...H...I...G. TFL1.ENMGT.VIEP..M...V...F..PTTKMN.S.N-KKQVS..H..F..S.SSK...IH.G...S.F...I...V.G..D.F.K.H...I..N..G. ZmTFL1.S----.SVEP...E...S.NPC.KMI...NSNKLVF..H.IY..AI.SK...Q.G...S.F...T...V.G..D.Y...H...I...G. OnFT Hd3a FT PnFT OnTFL1 OsCEN TFL1 ZmTFL1 B TTATFGTEIVCYESPRPSLGIHRFVFVLFHQLGRQTVYA-PGWRQNFNTRDFAELYNLGLPVAAVYFNCQREAGSGGRRMQD.A.S..Q.VM...TM...L...Q K...S...VYP.GT...N...N.S.TA...V..I..R E...I...FY...S.C...L--.G.S..H.T...N...TM...R V...S...S...R--.D.S..R.V.P...RI...M.KR.L..GP-.IS.DR...G...EHD...S...A...TAARR.----.D.S..R.V.S...NI...IL...R.KR..A.SP-.PS.DR.S..Q...DND...A...TAARR.----.D...K.V.S..L...I...R.KQ.RVIFPNIPS.DH...K..VE.D...F..A...TAARK.----.D.S..R.VIS...NI...I...K.K...TV-.SF.DH...Q...END...A...TAARR SaFT [Sinapis alba] FT [Arabidopsis thaliana] PcTFL1 [Pyrus communis] CiTFL1 [Citrus sinensis] PnTFL1 [Populus nigra] VvTFL1 [Vitis vinifera] CEN [Antirrhinum majus] CET [Nicotiana tabacum] CaSP [Capsicum annuum] LeSP [Lycopersicon esculentum] StTFL1 [Solanum tuberosum] OnTFL1 [Oncidium] OsCEN [Oryza sativa] FDR1 [Oryza sativa] ZmTFL1 [Zea mays] BnTFL1 [Brassica napus] TFL1 [Arabidopsis thaliana] OnFT [Oncidium] Hd3a [Oryza sativa] LpFT3 [Lolium perenne] AeFT [Aegilops tauschii] TaFT [Triticum aestivum] HvFT [Hordeum vulgare] PpFT [Prunus persica] MdFT [Malusxdomestica] PnFT [Populus nigra] CiFT [Citrus unshiu] Eudicots FT Eudicots TFL1 Monocots TFL1 Monocots FT Eudicots FT CmFTL1 [Cucurbita moschata] 918 CmFTL [Cucurbita moschata] Fig. 1 Sequence comparison of OnFT, OnTFL1 and related PEBP proteins. (A) Alignment of amino acid sequences of OnFT, OnTFL1 and related PEBP proteins such as FT and TFL1 (Arabidopsis), PnFT ( Populus nigra ), Hd3a and OsCEN (rice) and ZmTFL1 ( Zea mays ). The boxed region represents a highly conserved amino acid among PEBP proteins (His for TFL1 and Tyr for FT orthologs). Amino acid residues identical to OnFT in this alignment are indicated by dots. Dashes have been introduced to improve alignment. The arrowhead indicates the position of the introns. Continued 16

4 FT and TFL1 orthologs from Orchid A B C AB IF D E P PB F1 F F3 F F F6 Col Cal R L S P F Fig. Shoots and flowers for O. Gower Ramsey. (A) Close-up of the axillary bud region (AB) inside the vegetative shoot; R, roots. Bar = mm. (B) A young O. Gower Ramsey plant in the vegetative stage with 1 leaves. The number indicates the leaf position from top to bottom. Bar = 1 mm. (C) A mature O. Gower Ramsey plant in the reproductive stage produced the inflorescence (IF) from the axillary bud in the base of the third leaf. A mature pseudobulb (PB) was produced during this stage. The number indicates the leaf position from top to bottom. Bar = 3 mm. (D) Flower buds of O. Gower Ramsey at different developmental stages (F1 F6). Bar = mm. (E) An O. Gower Ramsey flower consisting of three sepals (S), two petals (P), a lip (L) with red-brown around the callus (Cal) and a reproductive organs column (Col). Sepals and petals are yellow with red brown bars and blotches toward the base of the segment. Bar = mm. (F) The reverse side of the O. Gower Ramsey flower. Three sepals (S) were observed. Bar = mm. (G) Close-up of the anther cap (arrowed), which covers the reproductive organs column (Col). Bar = 1 mm. (H) The pollinarium (male reproductive organ), which consists of two pollinia (po), a stalk (sk) and the brown viscidium (v). Bar =. mm. S S S G H Col v sk po Fig. 1 Continued The amino acid sequences were aligned by the BIOEDIT program ( using CLUSTALW MULTIPLE ALIGNMENT with default parameters. (B) Phylogenetic analysis of orthologs of the FT/TFL1 genes. Based on the amino acid sequence of the fulllength protein, OnFT was closely related to Hd3a, whereas OnTFL1 was most related to OsCEN. The names of the OnFT and OnTFL1 proteins are in bold and underlined. Names of the plant species for each FT/TFL1 gene are listed behind the protein names. Amino acid sequences of FT/TFL1 orthologs were retrieved via the NCBI server ( ). The tree was generated by the DNA Data Bank of Japan ( ), whereas the distance was calculated based on CLUSTALW using the phylogenetic tree software TreeView. Numbers on major branches indicate bootstrap percentages for 1 replicate analyses. 17

5 C.-J. Hou and C.-H. Yang A OnFT B OnFT C root axillary bud leaf 1leaf young stage vegetative stage leaf 3 leaf leaf pseudobulb leaf on bulb 1 leaf on bulb leaf on bulb 3 leaf on bulb leaf on bulb reproductive stage OnTFL1 root axillary bud leaf 1leaf leaf 3 lea fleaf pseudobulb leafonbulb 1 leafonbulb leafonbulb 3 leaf on bulb leaf on bulb for OnFT was significantly higher in axillary buds than in other organs tested ( Fig. 3A ). When the expression of OnFT in flowers was examined, OnFT mrna was detected in F1 F6 flower buds ( mm and 1 mm in length, Fig. D ) of different developmental stages ( Fig. 3B ). Interestingly, expression of OnFT was lower during flower maturation and was significantly higher in young F1 flower bud than in F6 mature flower bud ( Fig. 3B ). In addition, the expression level of OnFT was about six-fold higher in F1 flower buds than in vegetative axillary buds ( Fig. 3B ). When floral organs from mature D F1 F F F F F6 axilliarybuds lip sepal petal carpel stamen cap flowers were examined (Fig. E H), OnFT was expressed in sepal, petal, carpel and cap, but it was barely detected in lips and stamen ( Fig. 3B ). When the expression of OnTFL1 was analyzed during the vegetative stage, its mrna was detected only in axillary buds ( Fig. A ) and was absent in all other organs tested ( Fig. 3C ). OnTFL1 mrna was only detected in pseudobulb ( Fig. C ) during the reproductive stage ( Fig. 3C ). The expression level of OnTFL1 was relatively higher in pseudobulb than in axillary buds ( Fig. 3C ). In contrast to OnFT, the expression of F1 OnFT OnFT Fig. 3 Detection of expression of OnFT and OnTFL1 by real-time PCR. (A) and (C) Total RNA isolated from axillary bud, roots ( Fig. A ), five leaves of the vegetative stage ( Fig. B ), pseudobulb and five leaves of the reproductive stage ( Fig. C ) for O. Gower Ramsey were used as templates to detect the expression of OnFT (A) and OnTFL1 (C). (B) Total RNA isolated from flower buds of six different developmental stages, from sepal, petal, lip, stamen, carpel and cap of mature flowers, was used as templates to detect the expression of OnFT. (D) To examine whether the expression of OnFT was influenced by light, total RNA was isolated and analyzed from sepals and petals from O. Gower Ramsey every h. Sample collection started at the beginning of the light period (time ) and continued every h for h in LD conditions. Each experiment was repeated three times with similar results. 18

6 FT and TFL1 orthologs from Orchid OnTFL1 was completely absent from all the flower buds of different developmental stages and from all flower organs tested (data not shown). To explore whether the expression of OnFT and OnTFL1 was influenced by daily oscillation, mrna expression for OnFT and OnTFL1 was analyzed every h over a h period in long day (LD) conditions using quantitative real time PCR. The results indicated that OnFT mrna is expressed at its highest levels at 8 1 h of the light period and its lowest expression at dawn ( Fig. 3D ). These results suggest that OnFT of orchids is regulated by light similarly to FT in Arabidopsis. Differently from OnFT, expression of OnTFL1 was not regulated by the light and was detected at a similar level at most time points (data not shown). Ectopic expression of OnFT caused early flowering and partially restored ft-1 mutant phenotypes in Arabidopsis To explore whether OnFT from orchid is able to regulate flower transition, OnFT driven by the cauliflower mosaic virus (CaMV) 3S promoter was transformed into wild-type Arabidopsis plants for functional analysis. Thirty-three independent 3S:: OnFT transgenic Arabidopsis T 1 plants were obtained. Nine plants were phenotypically indistinguishable from untransformed wild-type plants, whereas the other plants showed identical phenotypes by flowering earlier than wild-type plants. These 3S:: OnFT transgenic plants (Fig. A ) flowered at about d after sowing by producing about six rosette leaves ( Table 1 ). The flowering time of wild-type Columbia plants was about 9 d by producing eight or nine rosette leaves ( Table 1 ). In contrast to wildtype plants, inflorescence was terminated by producing one or two flowers at the end of inflorescence in these 3S:: OnFT plants ( Fig. A, B ). To explore whether the early flowering phenotype was correlated with OnFT expression in 3S:: OnFT transgenic plants, real time PCR analysis was performed. As shown in Fig. A, higher OnFT expression was observed in the severe 3S:: OnFT transgenic plants than in the transgenic plants with the less severe or wild-type phenotype. Further analysis indicated that the promotion of flowering time in severe 3S:: OnFT transgenic plants was also correlated with significant up-regulation of the flower meristem identity gene AP1 in transgenic plants ( Fig. B ). To further confirm whether OnFT could compensate for the FT function in Arabidopsis, ectopic expression of OnFT was performed in Arabidopsis ft-1 mutants. Six independent 3S:: OnFT/ft-1 transgenic Arabidopsis plants were obtained. One transgenic plant was phenotypically indistinguishable from ft-1 mutants whereas the other five plants showed different degrees of flowering earlier than ft-1 mutants ( Fig. C, D ). The average flowering time for these 3S:: OnFT /ft-1 transgenic plants is about d after sowing by producing 18 rosette leaves ( Table 1 ). The flowering time of ft-1 mutants was about 61 d and more than 39 rosette leaves were produced ( Table 1 ). However, the average flowering time for these 3S:: OnFT/ft-1 transgenic plants was still about 8 d later than that of wild-type Landsberg plants ( Fig. D and Table 1 ). This result indicates that although the function of orchid OnFT is similar to that of Arabidopsis FT, it is still not able to completely complement Arabidopsis FT in regulating flowering time. To explore whether the early flowering phenotype in 3S:: OnFT/ft-1 transgenic plants correlated with OnFT expression in the transgenic plants, real time PCR analysis was performed. As shown in Fig. C, higher OnFT expression was observed in the severe phenotype of 3S:: OnFT/ft-1 transgenic plants compared with the transgenic plants with a phenotype less severe or indistinguishable from that of ft-1 plants. Further analysis indicated that the promotion of flowering time in severe 3S:: OnFT/ft-1 transgenic plants was also correlated with the significant up-regulation of AP1 in transgenic plants ( Fig. D ). This result clearly indicated that the phenotype generated in the 3S:: OnFT/ft-1 transgenic Arabidopsis was due to the ectopic expression of the orchid OnFT gene. Ectopic expression of OnTFL1 delayed flowering and restored tfl1-11 mutant phenotypes in Arabidopsis To explore whether OnTFL1 from orchid is able to regulate flower initiation, OnTFL1 driven by the CaMV 3S promoter was transformed into wild-type Arabidopsis and 1 independent 3S:: OnTFL1 transgenic plants were obtained. Two plants were phenotypically indistinguishable from untransformed wild-type plants, whereas the other 1 plants showed a different degree of flowering later than wild-type plants ( Fig. E ). These 3S:: OnTFL1 transgenic plants ( Fig. E ) flowered at about d after sowing by producing 19 rosette leaves ( Table 1 ). The flowering time of wild-type Columbia plants was about 9 d and fewer than nine rosette leaves were produced ( Table 1 ). In addition, extended lateral branching of curly leaves with reduced size and more shoots were produced in the severe 3S:: OnTFL1 transgenic plants during late developmental stage ( Fig. F, G). Further analysis indicated that the delay in flowering time in 3S:: OnTFL1 transgenic plants was correlated with high expression of OnTFL1 and down-regulation of AP1 ( Fig. 6A ). This result indicates that OnTFL1 acts similarly to TFL1 in controlling floral initiation. To further confirm whether OnTFL1 could compensate for TFL1 function in Arabidopsis, ectopic expression of OnTFL1 was performed in Arabidopsis tfl1-11 mutants. Eleven independent 3S:: OnTFL1/tfl1-11 transgenic Arabidopsis plants were obtained. Three plants were phenotypically indistinguishable from untransformed tfl1-11 mutant plants, whereas the other eight plants showed a different degree of flowering later than tfl1-11 mutant plants ( Fig. H 19

7 C.-J. Hou and C.-H. Yang A B C D F H WT 3S::OnFT-1-6 WT 3S::OnFT/ft-1-7 ft-1 WT G E 3S::OnTFL1 I 3S::OnFT/ft-1-7 3S::OnFT/ft-1-1 3S::OnFT/ft-1-9 WT 3S::OnTFL1-3S::OnTFL1-1 3S::OnTFL1-6 WT tfl1-11 3S::OnTFL1-H8Y-1-1 WT tfl1-11 3S::OnTFL1/tfl S::OnTFL1/tfl S::OnTFL1/tfl Fig. Phenotypic analysis of transgenic Arabidopsis plants that ectopically expressed OnFT, OnTFL1 or OnTFL1-H8Y. (A) A -day-old 3S:: OnFT plant 1-6 (right) grown on soil flowered at 17 d after germination and was much earlier than a wild-type plant (left). Inflorescence was terminated (arrowed) in this 3S:: OnFT plant. (B) Close-up of the determined inflorescences that terminated with two flowers at the end of the inflorescence of the 3S:: OnFT plant in (A). (C) Three -day-old 3S:: OnFT /ft-1 plants showed different flowering times. The earliest flowering plant 1-7 (left) flowered at d, the moderate early flowering plant 1-1 (middle) flowered at 9 d and the late flowering plant 1-9 (right) flowered at d after germination. (D) A 3S:: OnFT/ft-1 plant 1-7 (middle) grown on soil flowered earlier ( d) than a ft-1 mutant (6 d) (right) and still later than a wild-type Landsberg plant (left) (6 d). In this stage, ft-1 mutants did not flower and produced only rosette leaves. (E) Three -day-old 3S:: OnTFL1 plants showed different degree of flowering later than the wild-type Columbia plant (left), which flowered at 3 d after germination. Plant 1- (middle, left) showed slightly late flowering (3 d), plant 1-1 (middle, right) showed moderately late flowering (39 d) and plant 1-6 (right) showed the latest flowering time (8 d). (F) A -day-old 3S:: OnTFL1 plant (right) grown on soil flowered later than a wild-type plant (left). In this 3S:: OnTFL1 plant, abnormally small curly leaves and multiple branches were produced. (G) Close-up of the 3S:: OnTFL1 plant in (F). Extended lateral branching of curly leaves and more shoots (arrowed) were observed. (H) A 3S:: OnTFL1 /tfl1-11 plant 11-1 (right) grown on soil flowered later (8 d) than 11- (middle, right) (36 d), 11-7 (middle) (31 d), untransformed tfl1-11 (middle, left) ( d) and wild-type Columbia plant (left) (3 d). Plant 11-7 is phenotypically similar to tfl1-11 in flowering early and producing determinant inflorescences (arrowed). (I) A -day-old 3S:: OnTFL1-H8Y plant 1-1 (right) grown on soil flowered earlier than a wild-type Columbia plant (left) and similarly to a tfl1-11 mutant (middle). 1

8 FT and TFL1 orthologs from Orchid Table 1 Bolting time and leaf number of wild-type, ft-1, tfl1-11 mutant and transgenic Arabidopsis plants and ectopic expression of OnFT, OnTFL1 or OnTFL1-H8Y under LD (16 h L/8 h D) conditions Genotype No. of Days of Rosette Terminal plants flowering a leafb flower WT (L) c ± ±.93 ft-1 (L) ± ±. 3S:: OnFT/ft-1 (L) 6.33 ± ± 1.7 WT (C) ± ± S:: OnFT (C) ± 3.9. ± 1. + tfl1-11 (C) ±.9.31 ± S:: OnTFL1 (C) ± ± 6.7 3S:: OnTFL1/tfl1-11 (C) ± ± 3.1 3S:: OnTFL1-H8Y (C) 1.76 ±.1.81 ±.68 a Average number of days for plants to develop an elongated inflorescence with floral buds. b Average number of rosette leaves for plants at the time of floral bud emergence. c L, Landsberg background; C, Columbia background. and Table 1 ). These 3S:: OnTFL1/tfl1-11 transgenic plants flowered at about 3 d after sowing by producing about seven rosette leaves ( Table 1 ). The flowering time of the tfl1-11 mutants was about d and fewer than six rosette leaves were produced ( Table 1 ). In addition, the terminal flowers produced in tfl1-11 were not observed in these late flowering 3S:: OnTFL1/tfl1-11 plants (Fig. H). These results indicate that the function of orchid OnTFL1 is able to complement Arabidopsis TFL1 in controlling flowering initiation. Further analysis indicated that the delay in flowering time in 3S:: OnTFL1/tfl1-11 transgenic plants was correlated with high expression of OnTFL1 and down-regulation of AP1 ( Fig. 6B ) in an Arabidopsis tfl1-11 background. This result further supported that OnTFL1 acts similarly to TFL1 in controlling floral initiation. A single amino acid substitution converts the function of OnTFL1 in regulating flower transition It has been reported that the conserved amino acid His-88 in TFL1 plays an important role in the difference in function between TFL1 and FT. Substitution for the amino acid in this position (His to Tyr for TFL1) resulted in the functional conversion of TFL1 to FT in transgenic Arabidopsis ( Hanzawa et al. ). It is worthwhile to test whether the role of this amino acid is also conserved in other plant species. In orchid, the corresponding His-8 for OnTFL1 was substituted by Tyr to generate OnTFL1-H8Y, and this was ectopically expressed in Arabidopsis. Twenty-one independent 3S:: OnTFL1-H8Y transgenic plants were obtained and all showed identical novel phenotypes by flowering earlier than wild-type plants. These 3S:: OnTFL1-H8Y transgenic plants ( Fig. I ) flowered at about 6 d after sowing by producing five or six rosette leaves, similar to the flowering observed in tfl1-11 mutants ( Table 1 ). The flowering time of wild-type Columbia plants was about 9 d and more than eight rosette leaves were produced ( Table 1 ). Further analysis indicated that the promotion of flowering in these 3S:: OnTFL1-H8Y transgenic plants was due to significant up-regulation of AP1 ( Fig. 6C ). These results indicate that the substitution of His by Tyr at position 8 of orchid OnTFL1 is sufficient to convert the function of OnTFL1 from delaying to promoting flowering, as OnFT does. Discussion In this study, to investigate the role of the PEBP/RKIP gene family in regulating the transition from vegetative to reproductive growth in the orchid O. Gower Ramsey, orthologs for PEBP were identified and characterized in O. Gower Ramsey. OnFT and OnTFL1, the orchid genes characterized here, are closely related to FT and TFL1 genes based on their protein sequences ( Fig. 1 ). This suggests that OnFT and OnTFL1 are potentially FT and TFL1 orthologs that regulate flower transition and initiation in O. Gower Ramsey. The interesting characteristics of OnFT are its spatial and temporal expression patterns. FT was specifically induced by CO in phloem cells, but its mrna can be detected in leaves ( Takada and Goto 3, An et al. ). A very low accumulation of Hd3a mrna was detected in leaf blade tissue and sheath in rice ( Tamaki et al. 7, Wu et al. 8 ). The expression of OnFT was also relatively low in leaves during the vegetative stage and increased during the reproductive stage. Interestingly, OnFT was highly expressed in the axillary buds during the vegetative stage. The axillary buds of orchid contain differentiated tissue ( Fig. A ) and can potentially be converted into inflorescences during flower transition. These results indicate that the high expression of OnFT in the axillary buds during the early vegetative stage is not sufficient for the transition to reproductive development in O. Gower Ramsey. These results also suggest an important role for the production of pseudobulb before flower transition in O. Gower Ramsey. The pseudobulb ( Fig. C ) is an essential organ for Oncidium, involved in the storage of nutrition and the supply of carbohydrate, minerals and water for axillary buds and inflorescence development ( Tan et al. ). In the absence of the pseudobulb, the inflorescence is not able to develop from the axillary buds, even with high OnFT expression. It is interesting to notice that expression of OnFT was relatively lower in the vegetative leaves before the formation of pseudobulb than in the leaves in reproductive stage ( Fig. 3A ). This indicated that formation of the pseudobulb in O. Gower Ramsey may need low expression of OnFT. Interestingly, a similar bud set in the fall correlated with the down-regulation of PtFT expression has been reported in the aspen tree P. trichocarpa ( Böhlenius et al. 6 ). 11

9 C.-J. Hou and C.-H. Yang A B 6 OnFT Columbia Columbia 3S::OnFT-1-1 3S::OnFT-1-1 3S::OnFT-1-7 3S::OnFT-1-7 3S::OnFT-1-6 3S::OnFT-1-6 AP1 When the expression of OnFT in flowers was examined, OnFT mrna was detected strongly in flower buds of different developmental stages ( Fig. 3B ), similar to observations for FT orthologs ( Carmona et al. 7, Igasaki et al. 8 ). However, expression of OnFT was significantly higher in young F1 flower buds and gradually decreased during flower maturation ( Fig. 3B ). This pattern was different from that of FT expression, which in contrast, gradually increased during flower maturation ( Kobayashi et al ). Furthermore, similarly to FT orthologs ( Suárez-López et al. 1, Yanovsky and Kay, Böhlenius et al. 6, Hsu C D Landsberg Landsberg OnFT ft-1 3S::OnFT/ft-1-7 3S::OnFT/ft-1-1 3S::OnFT/ft-1-9 AP1 ft-1 3S::OnFT/ft-1-7 3S::OnFT/ft-1-1 3S::OnFT/ft-1-9 Fig. Detection of OnFT and AP1 expression by real-time PCR in transgenic Arabidopsis plants that ectopically expressed OnFT. (A, B) Total RNA isolated from three 3S:: OnFT transgenic Arabidopsis and from one untransformed wild-type Columbia plant was used as template to detect the expression of OnFT (A) and AP1 (B). Plant 1-6 (Fig. A) showed the earliest flowering (17 d) and 1-7 showed early flowering ( d), whereas 1-1 (8 d) was like wild-type 3S:: OnFT transgenic plants. Both OnFT (A) and AP1 (B) expression were clearly higher in 1-6 than in 1-7 and 1-1. OnFT expression was undetectable in untransformed wild-type plants. (C, D) Total RNA isolated from three 3S:: OnFT/ft-1 transgenic Arabidopsis (1-7, 1-1 and 1-9), one untransformed wild-type Landsberg plant and one ft-1 mutant was used as template to detect the expression of OnFT (C) and AP1 (D). Plant 1-7 flowered earlier ( d) than 1-1 (9 d) and 1-9 ( d) (Fig. C). Flowering occurred earlier and OnFT expression was higher in 1-7 than in 1-1 and 1-9 plants (C). AP1 expression was clearly up-regulated in 1-7 compared with 1-1, 1-9 and ft-1 mutants (D). et al. 6, Gyllenstrand et al. 7, Hayama et al. 7 ), OnFT mrna is also regulated by light with the level highest at the 8th to 1th hour of the light period and lowest at dawn. This pattern is, however, slightly different from FT of Arabidopsis, which has its level highest at the 16th hour of the light period and the lowest at dawn under LD conditions ( Yamaguchi et al., Corbesier et al. 7, Fujiwara et al. 8 ), or Hd3a of rice and Pn FT of Pharbitis, which have their highest level at dawn under SD conditions ( Izawa et al., Ishikawa et al., Hayama et al. 7, Tamaki et al. 7 ). The difference in the expression pattern for FT orthologs in 1

10 FT and TFL1 orthologs from Orchid A B OnTFL1 AP1 18 3S::OnTFL1-1- 3S::OnTFL S::OnTFL OnTFL1 AP1 3S::OnTFL1/tfl S::OnTFL1/tfl S::OnTFL1/tfl C Columbia AP1 3S::OnTFL1-1- 3S::OnTFL S::OnTFL S::OnTFL1/tfl S::OnTFL1/tfl S::OnTFL1/tfl tfl1-11 3S::OnTFL S::OnTFL1/tfl S::OnTFL1-H8Y-1-1 Fig. 6 Detection of OnTFL1 and AP1 expression by real-time PCR in transgenic Arabidopsis plants that ectopically expressed OnTFL1. (A) Total RNA isolated from three 3S:: OnTFL1 (1-, 1-1, 1-6) shown in Fig. E was used as template to detect the expression for OnTFL1 and AP1. OnTFL1 expression was higher in 1-6 than in 1-1 and 1-. By contrast, AP1 expression was significantly lower in 1-6 than in 1-1 and response to light may be due to the fact that Oncidium is a light-neutral plant ( Hew and Yong 1997 ), whereas Arabidopsis is an LD plant and rice is an SD plant. The function of OnFT in flower transition was further supported by functional complementation analysis. The earlyflowering phenotype and the loss of inflorescence indeterminacy observed in 3S:: OnFT transgenic plants ( Fig. A, B ) were no doubt similar to the phenotypes observed in Arabidopsis or various other species that ectopically express FT orthologs ( Kardailsky et al. 1999, Kobayashi et al. 1999, Lifschitz et al. 6, Hayama et al. 7, Igasaki et al. 8, Komiya et al. 8, Takahashi et al. 9 ). Interestingly, the expression of AP1, a marker for floral initiation and the downstream gene of FT, was up-regulated in 3S:: OnFT transgenic Arabidopsis plants and the level of the AP1 transcripts was strongly correlated with the expression of OnFT ( Fig. ). This indicates that constitutive expression of OnFT acts similarly to Arabidopsis FT in regulating flower transition by activating AP1. In addition, when OnFT was ectopically expressed in Arabidopsis ft-1 mutants, the restoration of the mutant phenotype was observed in transgenic plants by promoting flowering in the ft-1 mutants ( Fig. C, D ). Interestingly, although they flowered earlier than ft-1 mutants, these 3S::OnFT/ft-1 transgenic plants still flowered later than wild-type plants ( Fig. D and Table 1 ). This indicates that the function of orchid s FT ortholog, OnFT, was not able to completely complement Arabidopsis FT in regulating flowering time. In contrast to OnFT, the expression of OnTFL1 transcripts was detected only in axillary bud during the vegetative stage and in the pseudobulb of the reproductive phase ( Fig. 3C ). In flowers, the expression of OnTFL1 was completely absent. This is not surprising since the axillary buds of orchid can be thought of as an inflorescence meristem region that is converted into an inflorescence during flower transition. High OnTFL1 expression in axillary bud could repress the flower transition during the vegetative stage. This is similar to the 1-. The flowering times for 1-6, 1-1 and 1- were 8, 39 and 3 d after germination, respectively. (B) Total RNA isolated from three 3S:: OnTFL1/tfl1-11 (11-7, 11-, 11-1) shown in Fig. H was used as template to detect expression of OnTFL1 and AP1. OnTFL1 expression was higher in 11-1 and 11- than in By contrast, AP1 expression was clearly lower in 11-1 and 11- than in The flowering times for 11-1, 11-1 and 11-7 were 8, 36 and 31 d after germination, respectively. (C) Detection of AP1 expression in 3S:: OnTFL1 (1-6), 3S::OnTFL1/tfl1-11 (11-1), 3S::OnTFL1-H8Y (1-1) transgenic Arabidopsis, untransformed wild-type Columbia plant and tfl1-11 mutant. AP1 was slightly up-regulated in tfl 1-11 mutants, downregulated in 3S:: OnTFL1 (1-6), 3S::OnTFL1/tfl1-11 (11-1) and significantly up-regulated in 3S::OnTFL1-H8Y (1-1) transgenic Arabidopsis. 13

11 C.-J. Hou and C.-H. Yang expression of TFL1 orthologs in shoot apical meristem that prohibit flower transition by restricting AP1 expression ( Weigel et al. 199, Bowman et al. 1993, Conti and Bradley 7 ). The high expression of OnTFL1 in pseudobulb strongly demonstrates that OnTFL1 plays an important role in controlling the length of the vegetative phase since the complete development of a pseudobulb to increase the storage of nutrition for the development of reproductive organs is essential for Oncidium ( Tan et al. ). Similarly to TFL1 of Arabidopsis, the expression of OnTFL1 was not regulated by light and was detected at a similar level at most time points. The function of OnTFL1 in flower transition was also revealed by functional complementation analysis. The delay of flowering and the production of more rosette leaves observed in 3S:: OnTFL1 transgenic plants ( Fig. E, F and Table 1 ) were similar to that observed in Arabidopsis or other species that ectopically express TFL1 orthologs ( Jensen et al. 1, Nakagawa et al., Pillitteri et al., Zhang et al., Böhlenius et al. 6, Mimida et al. 9 ). In addition, alteration of leaf and shoot morphology was observed in the severe 3S:: OnTFL1 transgenic plants ( Fig. F, G). Similar alteration of the architecture of the shoot by producing extended lateral branching leaves has been reported in plants that ectopically express LpTFL1 and IbTFL1 (Jensen et al. 1, Ordidge et al. ). When OnTFL1 was further ectopically expressed in Arabidopsis tfl1-11 mutants, the determinate inflorescence phenotype was restored, more leaves were produced and the flowering time was delayed when compared with the tfl1-11 mutant ( Fig. H and Table 1 ). Interestingly, expression of AP1 was down-regulated in both severe 3S:: OnTFL1 and 3S:: OnTFL1/tfl1-11 transgenic Arabidopsis plants ( Fig. 6C ). This indicates that constitutive expression of OnTFL1 acts similarly to Arabidopsis TFL1 in prohibiting flower transition and the development of floral meristem by negatively regulating AP1 ( Conti and Bradley 7 ). This result indicates that constitutive expression of OnTFL1 is sufficient to act as a parallel of TFL1 in transgenic Arabidopsis plants. Since a conserved amino acid in the ligand binding pocket (His-88 for TFL1 and Tyr-8 for FT) has been shown to play a critical role in determining the function of FT and TFL1 ( Hanzawa et al. ), a single amino acid for the corresponding His residue of OnTFL1 (His-8) was substituted by Tyr to generate OnTFL1-H8Y, and this was ectopically expressed in Arabidopsis. Interestingly, an early flowering phenotype was observed in these 3S:: OnTFL1-H8Y plants ( Fig. I ). Furthermore, the expression of AP1 was significantly up-regulated in these 3S:: OnTFL1-H8Y transgenic Arabidopsis plants ( Fig. 6C ). This indicates that only an amino acid change in OnTFL1-H8Y was sufficient to convert the function of OnTFL1 into OnFT, a similar result to that observed only for FT/TFL1 in Arabidopsis. Therefore, the importance of this pocket binding residue (His for TFL1 and Tyr for FT) for affecting biochemical action and producing dramatically reversed effects in the inflorescence initiation of FT and TFL1 during evolution was confirmed through our investigation in a diverse orchid plant. In summary, two putative PEBP genes, OnFT and OnTFL1, that regulate inflorescence initiation were characterized from O. Gower Ramsey. The similarity of expression patterns between OnFT or OnTFL1 and their orthologs indicates that their function is highly conserved during evolution. This assumption is further supported by the alteration of floral initiation and AP1 expression in transgenic plants that ectopically express these two genes. Furthermore, the ability of OnTFL1-H8Y to function as FT supported the idea that the conservation of the amino acid identified in the ligandbinding pocket correlated with the function of the FT/TFL1 proteins in plants. The characteristics of the two orchid genes obtained in this study provide useful information for the understanding of the relationships between the PEBP genes in regulating flower transition. Materials and Methods Plant materials and growth conditions Plants of O. Gower Ramsey used in this study were grown and maintained in the greenhouse of National Chung-Hsing University, Taichung, Taiwan. Arabidopsis ft-1 and tfl1-11 mutant lines were obtained from the Arabidopsis Biological Resource Center (Ohio State University, Columbia, OH, USA). Seeds for Arabidopsis were sterilized and placed on agar plates containing 1/ Murashige and Skoog medium (Murashige and Skoog 196) at C for d. The seedlings were then grown in a growth chamber under LD conditions (16-h light/8-h dark) at C for 1 d before being transplanted to soil. The light intensity of the growth chambers was 1 µe m s 1. Cloning of cdnas for OnFT and OnTFL1 from O. Gower Ramsey Total RNA extracted from - to -mm-long floral bud of Oncidium was used for cdna synthesis as described by Tzeng and Yang (1). Synthesized cdna fragments and FT /TFL1 degenerated primers were used in PCR experiments by the Touchdown program. Degenerated primers for OnFT : forward primer ( -ATGGTGGATCCNGAYGYNCCNAGY CC-3 ) and reverse primer ( -GTGYTGAAGTTCTGRCGCCA CCCNGG-3 ). Degenerated primers for OnTFL1 : forward primer ( -ACTGGATTGTCACAGACATTCCA-3 ) and reverse primer ( -TGAAGAAAACAGCDGCAACAGG-3 ). The amplified fragments containing partial sequence of OnFT and OnTFL1 that showed high sequence identity to PEBP genes were identified. Internal gene-specific primers for OnFT or OnTFL1 were designed for and 3 rapid amplified cdna ends ( - and 3 -RACE) by SMART RACE cdna amplification kit 1

12 FT and TFL1 orthologs from Orchid (BD Biosciences Clontech, Palo Alto, CA, USA). Gene-specific primers for and 3 -RACE of OnFT : -TCTCATTTTATT CCCAACGCTG-3 and -CTTAAGCCTTCAGTTGTAGTGG AGCAGCC-3. Gene-specific primers for and 3 RACE of OnTFL1 : -AACAGCGGCAACAGGAAGGCCCA-3 and - GGTTCCTTATGAGAGTCCGAGGCCGAG-3. The cdnas for OnFT were obtained by PCR amplification using the forward primer ( -CG GGATCC ATGAATAGAGAGAGAGACT CTTT-3 ) and reverse primer ( -CGGGATCCTCAATCCTG CATCCTTCTTCC-3 ). The cdnas for OnTFL1 were obtained by PCR amplification using the forward primer ( - CGGGATCCAGTACGCGGGGATAAGAAGC-3 ) and reverse primer ( -CGGGATCCTGACAACACAGACCCGTGC-3 ). The forward and reverse primers for OnFT and OnTFL1 contained the generated Bam HI recognition site ( -GGATCC-3, underlined) to facilitate the cloning of the cdnas. Generation of cdna with amino acid substitution for OnTFL1 To generate OnTFL1-H8Y, point mutagenesis was performed as described by the megaprimer PCR method ( Ke and Madison 1997 ). The forward primer ( -CCTGAGAGA A TACGTTCACTG-3 ) contained a mismatched base pair (from C to T, bold type and underlined) was designed and used in PCR with reverse primer ( -CGGGATCCTGACAA CACAGACCCGTGC-3 ) to generate the site-directed mismatch DNA fragment (with His substituted by Tyr at position 8). This DNA fragment was then used as a reversed megaprimer in the second round of PCR with forward primer ( -CGGGATCCAGTACGCGGGGATAAGAAGC-3 ) to generate cdna of OnTFL1-H8Y. Real-time PCR analysis Quantitative real-time PCR was carried out using Mini Opticon (Bio-Rad Laboratories, Hercules, CA, USA) and SYBR Green Master Mix (Toyobo, Osaka, Japan) for time-course ( h) transcript measurements. The amplification condition was 9 C for 1 min, followed by cycles of amplification (9 C for 1 s, 8 C for 1 s, 7 C for 3 s) and plate reading after each cycle. Primers used for quantitative real-time PCR for OnFT : forward primer ( -ACCTCAGGACTTTCTACA CTCTTG-3 ) and reverse primer ( -GAAACAGCACGAACA CGAAGC-3 ), for OnTFL1 : forward primer ( -TTGTAGTTGG TAGAGTTATAGGAGAAG-3 ) and reverse primer (-AT CAGTCATAATCAGTGTGAAGAAAG-3 ), for AP1 : forward primer ( -CTAAAACCACTCTTACCCAAATCTCTC-3 ) and reverse primer ( -TGTCACTTGTCTATTGATCTTGTTCT C-3 ). The O. Gower Ramsey α-tubulin gene and the Arabidopsis UBQ1 gene were used as normalization control for orchid tissue and Arabidopsis tissue, respectively. For O. Gower Ramsey α-tubulin : forward primer ( -GG ATTAGGCTCTCTGCTGTTGG-3 ) and reverse primer ( - GTGTGGATAAGACGCTGTTGTATG-3 ), for Arabidopsis UBQ1 : forward primer ( -CTCAGGCTCCGTGGTGG TATG-3 ) and reverse primer ( -GTGATAGTTTTCCCAG TCAACGTC-3 ). Data were analyzed using Gene Expression Macro software (version 1.1, Bio-Rad). Plant transformation and transgenic plant analysis A Bam HI fragment containing the full-length cdna for OnFT or OnTFL1 was cloned into the binary vector pbi11 (BD Biosciences Clontech) under the control of the CaMV 3S promoter. The sense constructs were orientation determinant by PCR and used for further plant transformation. Arabidopsis plants were transformed through a floral dip method as described elsewhere ( Clough and Bent 1998 ). Transformants that survived in medium containing kanamycin ( µg ml 1 ) were further verified by RT PCR analyses. Acknowledgments This work was supported by grants to C.-H. Y. from National Science Council, Taiwan, ROC, grant numbers: NSC B--6 and NSC B--19. This work was also supported by Y/B grant from the Ministry of Education. References Abe, M., Kobayashi, Y., Yamamoto, S., Daimon, Y., Yamaguchi, A., Ikeda, Y., et al. 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