(Dendrobium Crumenatum Swartz) and Its Expression Under Different Photoperiod Conditions

The Horticulture Journal 86 (2): 252–262. 2017. e Japanese Society for doi: 10.2503/hortj.MI-123 JSHS Horticultural Science http://www.jshs.jp/

Characterization of a CONSTANS-like Gene from Pigeon Orchid (Dendrobium crumenatum Swartz) and its Expression under Different Photoperiod Conditions

Wanita Kaewphalug1, Pattana Srifah Huehne1,2,3 and Ajaraporn Sriboonlert1,2*

1Department of Genetics, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand 2Centre for Advanced Studies in Tropical Natural Resources, Kasetsart University, Bangkok 10900, Thailand 3Laboratory of Biotechnology, Chulabhorn Research Institute, Bangkok 10210, Thailand

Orchids are economically valuable as cut flowers and in pot plant markets. However, a juvenility phase that is too long is the main disadvantage for commercial orchids. To understand the gene involving floral transition controls in orchids, a CONSTANS-like (COL) gene in the photoperiodic flowering pathway was isolated from Dendrobium crumenatum (pigeon orchid). The cDNA isolated has an open reading frame (ORF) of 969 bp, encoding 322 amino acids. Sequence alignment based on amino acid sequences revealed that the Dendrobium crumenatum COL (DcCOL) shared a high identity with COL isolated from other plant species including Phalaenopsis COL (85%), Oryza sativa Hd1 (70%), Erycina pusilla COL5 (EpCOL5) (66%), and Arabidopsis thaliana CO (39%). DcCOL has three conserved domains (CCT, B-box I, and B-box II domains) and is classified in group I CO/COL by phylogenetic analysis in the Arabidopsis B-box zinc finger protein family. Quantitative real-time RT-PCR demonstrated DcCOL was expressed in all stages of development and all tissue types with the highest expression in floral buds and opened flowers of mature orchids. The expression pattern under photoperiod pathway demonstrated a diurnal expression. The DcCOL was accumulated in the dark in all photoperiodic conditions, i.e., long, neutral, and short days suggesting that the regulation of DcCOL was controlled in a circadian rhythm-dependent manner. The results suggested that photoperiodism is not the main factor in D. crumenatum floral induction control. This DcCOL expression pattern coincided with the D. crumenatum flowering behavior in which the flowering occurs before dawn and lasts for only 24 h implying the function of DcCOL is related to flowering.

Key Words: floral transition, photoperiodic, reproductive, vegetative.

juvenility, polyploidy, a large genome size, and a high Introduction chromosome number (Pellegrino et al., 2009; Russell Orchidaceae is the largest family of angiosperms et al., 2010). One of the main challenges in orchid with the number of species exceeding 25000 (Atwood, research and orchid breeding programs is their pro- 1986). The members of this plant family have high di- longed vegetative phase (Yu and Goh, 2001). A better versity in both vegetative and floral morphologies (da understanding of floral transition controls in orchids Silva et al., 2014; Xu et al., 2006). Many orchid genera would enable shorter juvenility periods and facilitate are economically valuable, namely Phalaenopsis (Fu enhanced orchid breeding programs. et al., 2011; Hsiao et al., 2011; Su et al., 2011), Dendrobium crumenatum Swartz (pigeon orchid) is a Cymbidium (Wu et al., 2009), Oncidium (Chang et al., member of the Dendrobium genus that can flower more 2011), and Dendrobium (da Silva et al., 2014). Howev- than once a year (Holttum, 1949; Meesawat and er, these commercial orchids have characteristics that Kanchanapoom, 2007). This pigeon orchid has unique are disadvantageous to genomic studies, including long features such as white flowers and fragrant and gregarious flowering (Holttum, 1949; Meesawat and Kanchanapoom, 2007). Its flowering cycles can be trig- Received; October 19, 2015. Accepted; July 5, 2016. First Published Online in J-STAGE on August 25, 2016. gered throughout the year, but its flower is short-lived * Corresponding author (E-mail: [email protected]). (Beaman et al., 2001; Yap et al., 2008). In natural

© 2017 The Japanese Society for Horticultural Science (JSHS), All rights reserved. Hort. J. 86 (2): 252–262. 2017. 253 conditions, the flowering of this orchid is induced by a (B-box I and B-box II) and a CCT domain, Group II prolonged cool period, i.e., heavy rainfall (Arditti, contains B-box I, high variation B-box II, and a CCT 1979; Holttum, 1949). Full flower opening is observed domain; Group III contains only one B-box domain (B- before dawn approximately nine days after cold induc- box I) and a CCT domain, Group IV contains B-box I tion, and the flowers last for only 24 h (Yap et al., and B-box II but no CCT domain, Group V contains 2008). The Dendrobium orchid species is well studied only a single B-box I domain (Gangappa and Botto, in floral developmental pathways. However, the molec- 2014; Khanna et al., 2009). These BBX transcription ular mechanism underlying the floral transition process factors have various functions involving developmental in this orchid is still unknown (Xu et al., 2006; Yap and growth regulation including seedling photomorpho- et al., 2008). genesis, photoperiodic regulation of flowering, shade Floral transition, described as the transition switch avoidance, and responses to biotic and abiotic stresses from the vegetative to the reproductive phase is impor- (Gangappa and Botto, 2014). CO and COL genes have tant for flowering plants (Boss et al., 2004). This been studied in various plant species, i.e., A. thaliana change from vegetative to reproductive growth is a crit- (Robson et al., 2001), barley (Griffiths et al., 2003), ical developmental process in a plant’s life cycle. The wheat (Nemoto et al., 2003), and sugar beet (Chia et al., transition is controlled by both endogenous signals and 2008). environmental stimuli. In Arabidopsis thaliana, four To date, 854 COL genes from 81 species have been major pathways of floral transition, including photoper- deposited in the PlantTFDB database (Liu et al., 2015). iod, autonomous, hormonal, and vernalization pathways COL genes have been identified in three orchid species, were identified (Borner et al., 2000; Mouradov et al., Erycina pusilla (Chou et al., 2013), Phalaenopsis 2002; Xiang et al., 2012). These processes are inte- hybrida (Zhang et al., 2011), and Oncidium ‘Gower grated by the function of two central flowering regula- Ramsey’ (Chang et al., 2011). In Oncidium ‘Gower tors, CONSTANS (CO) and FLOWERING LOCUS C Ramsey’, seven putative COLs were identified through (FLC). In the photoperiod pathway, light signals are 454 GS-FLX Pyrosequencing (Chang et al., 2011). In perceived by photoreceptors, which process the physi- Erycina pusilla, 12 EpCOL were identified through Il- cal signals. These photoreceptors signal GIGANTEA lumina sequencing. These EpCOL genes were divided (GI) gene and then trigger the CO gene (Fowler et al., into four groups (I–IV) according to their structural do- 1999; Hicks et al., 2001; Simpson and Dean, 2002; mains (Chou et al., 2013). Another COL from an orchid Suarez-Lopez et al., 2001; Zhang et al., 2011). CO is a that has been identified was Phalaenopsis hybrida COL transcription factor that promotes flowering by directly (PhalCOL). This PhalCOL was classified in the group I inducing expression of FLOWERING LOCUS T (FT) COL which contains two conserved B-box domain and and indirectly induces expression of SUPPRESSOR OF one CCT domain. Overexpression of the PhalCOL in OVEREXPRESSION OF CO1 (SOC1) (Komeda, 2004; tobacco demonstrated the early flowering phenotype Samach et al., 2000; Tiwari et al., 2010; Zhang et al., (Zhang et al., 2011). It is clear that a number of COLs 2011). This CO transcription factor contains three con- are present in plant genomes and each of them have dif- serve domains: two B-box domains and one CCT (CO, ferent functions, although next generation sequencing CO-like, TOC1) domain (Robson et al., 2001). The two technologies are used for identification of all the COLs B-box domains are a class of zinc-finger transcription in plants, traditional methods of cDNA amplification factor and are located near the amino terminus (N- and cloning are required for characterization of these terminus). These B-box domains have a function in genes. protein-protein interaction and transcription regulation One of the main floral induction factors in various (Chou et al., 2013; Gangappa and Botto, 2014; Suarez- Dendrobium orchids, including D. crumenatum, is Lopez et al., 2001; Thomas, 2006). The CCT domain known to be cold treatment. However, another factor locates near carboxyl terminus (C-terminus) which has that is required for floral transition in many flowering a function in protein-protein interactions, transcriptional plants is photoperiod. To examine whether photoperiod- regulation, and protein transport (Chou et al., 2013; ism plays a role in floral induction in this tropical Gangappa and Botto, 2014; Putterill et al., 1995; orchid, a CO/COL gene known to have function in flo- Robson et al., 2001; Strayer et al., 2000). The CCT do- ral transition via the photoperiodic pathway was iso- main of Arabidopsis CO was demonstrated to bind di- lated and characterized. The expression of this rectly to the FT promoter (Gangappa and Botto, 2014; D. crumenatum CO/COL gene was investigated under Tiwari et al., 2010). In Arabidopsis, seventeen members different day length conditions. The results provided of the CO gene family have been identified including here will improve our understanding of the regulation one CO and sixteen CO-like (COL) genes (Robson of orchid flowering through photoperiods and contrib- et al., 2001; Zhang et al., 2011). The Arabidopsis B-box ute knowledge for further analysis of flowering control zinc finger protein family (BBX) was classified into in Dendrobium orchids. five groups based on phylogenetic analysis and structure classification: Group I contains two B-box domains 254 W. Kaewphalug, P. Srifah Huehne and A. Sriboonlert

was cut and purified with a Gel/PCR DNA Fragments Materials and Methods Extraction Kit (FAVOGEN, Taiwan). A purified frag- Plant materials ment 75 ng was cloned into pGEM-T Easy vector 25 ng Dendrobium crumenatum plants were maintained (Promega, USA) as described in the manufacturer’s under natural light in a greenhouse at Kasetsart protocol and transformed into DH5α Escherichia coli University. The temperature of the greenhouse was ap- by a heat shock technique. Positive clones were se- proximately 30–35°C during the day time and 28–32°C lected on LB agar media containing ampicillin, isopro- during the night time throughout the year. The repro- pyl β-D-1-thiogalactopyranoside (IPTG), and X-gal and ductive stage of the plant was determined by the pres- further verified using colony PCR with GoTaq® Green ence of a leafless inflorescence stem produced at the (Promega). The positive clones were then sent for se- distal part of the pseudobulb (Fig. 4C). In the photo- quencing (Macrogen, South Korea). Full-length DcCOL period experiment, D. crumenatum specimens that was amplified using a SMARTer RACE cDNA Ampli- contained inflorescence stems were transferred into a fication kit (Clontech, USA). Both 3' and 5' RACE were growth chamber at a photosynthetic photon flux density performed in 5 μL reactions with total RNA of 0.5 μg, (PPFD) of 26 μmol·m−2·s−1. All plants were treated 1.2 mM 3' RACE CDS primer A for 3' RACE and with a neutral day condition (12 h light, 32°C and 12 h 1.2 μM 5'-CDS Primer A for 5' RACE. Nuclease-free dark, 30°C) for three days. The plants were then sepa- water was added to 2.38 μL for 3' RACE and 1.38 μL rated into three sets to be treated with long day, neutral for 5' RACE. The reactions were incubated at 72°C for day, and short day conditions for two days before the 3 min and chilled at 42°C for 2 min. DTT 4 mM was leaves were collected for quantitative RT-PCR analysis. then added to the reaction followed by 2 mM dNTP In the long day condition, plants were exposed to 16 h mix, 2x first strand buffer, RNase inhibitor 5.2 U, and light, 32°C and 8 h dark, 30°C. In the neutral condition, SMARTScribe Reverse Transcriptase 50 U. SMARTer plants were exposed to 12 h light, 32°C and 12 h dark, II A oligo 1.2 μM was only added to the 5' RACE reac- 30°C. In short day condition, plants were exposed to 8 h tion. Both reactions were then incubated at 42°C for light, 32°C and 16 h dark, 30°C. In all conditions, one 90 min and 70°C for 10 min. PCR was then performed leaf (1 × 2 cm) per plant was harvested every four hours in a 25 μL reaction comprised of 1.25 μL of either 3' or for two consecutive days. The harvested leaves were 5' RACE cDNA, 1x Advantage 2 PCR Buffer, 2 mM immediately submerged in liquid nitrogen and stored at dNTP Mix, 25x Advantage 2 Polymerase Mix, 0.2 μM −80°C. 10X Universal Primer A Mix (UPM) Long, and 5 mM primers. For 3' RACE, a W2-CO-F1 (5'-GTCTGGCTC CONSTANS-like cloning from D. crumenatum TGCGAGGTGTG-3') primer was used under the fol- Total RNA samples were extracted from leaves of lowing thermal cycling conditions: initial denaturation reproductive stage plants using TRIzol (Invitrogen, for 5 min at 94°C, 40 amplification cycles of denatura- USA) according to the manufacturer’s manual. tion for 1 min at 94°C, annealing for 30 s at 60°C, ex- D. crumenatum CO-Like (DcCOL) cDNA was synthe- tension for 1 min at 72°C, and final extension for 5 min sized using Viva 2-steps RT-PCR Kit (Vivantis, at 72°C. For 5' RACE-PCR, W2-CO-R1 (5'-ATGAA Malaysia). The PCR reaction included 1 μg of total AGCAGCGGTGGCG-3') primer was used under the RNA, 40 mM Oligo d(T)18 primer, 10 mM dNTPs. following thermal cycling condition: five amplification Water was added to 10 μL and incubated at 65°C for cycles of 30 s at 94°C and 72°C 3 min, five amplifica- 5 min. The reaction was placed on ice for 2 min. Later, tion cycles of denaturation for 30 s at 94°C, annealing 1x buffer M-MuLV and M-MuLV reverse transcriptase for 30 s at 68°C and extension for 3 min at 72°C and 25 100 U were added to the reaction and the volume was amplification cycles of denaturation for 30 s at 94°C, adjusted to 20 μL with water and incubated at 42°C for annealing for 30 s at 68°C and extension for 3 min at 90 min and 70°C 10 min. PCR was then performed in 72°C. PCR products from 5' and 3' RACE-PCR were the reaction containing 1 μL of cDNA, 5 μM of each checked on 1% agarose gel. The amplified sequences specific primers, W2-CO-F1 (5'-GTCTGGCTCTGC were cloned and sequenced as mentioned above. DNA GAGGTGTG-3') and W2-CO-R1 (5'-ATGAAAGC sequences from 3' and 5' RACE were blasted against AGCGGTGGCG-3'), 0.1 mM dNTPs, 1x Vi buffer, and the GenBank database using BLAST tools (Johnson 2.5 U Taq polymerase under the following conditions: et al., 2008). The 3' and 5' DNA sequences were then initial denaturation for 5 min at 94°C, 40 amplification assembled using CAP3 (Huang and Madan, 1999). cycles of denaturation for 1 min at 94°C, annealing for 30 s at 60°C, extension for 1 min at 72°C, and final ex- Protein sequence analysis tension for 5 min at 72°C. The W2-CO-F1 and W2-CO- The nucleotide sequence of COL from R1 primers were designed from the conserved regions D. crumenatum was translated by the ExPASy transla- of Dendrobium loddigesii (GU301089.1) and tion tool (Gasteiger et al., 2003). The translated protein Phalaenopsis hybrid (FJ469986.1) COL genes retrieved was blasted against Genbank database using blastp from Genbank database. The amplified cDNA fragment (Johnson et al., 2008). Conserved domains of DcCOL Hort. J. 86 (2): 252–262. 2017. 255 were identified using multiple sequence alignment. ATCAGCAAGCTGCTTGCCTGCAT-3') primers. Amino acid sequences of DcCOL and COL from other DcCOL gene product was detected using W3-CO-F (5'- plant species i.e. Dendrobium loddigisii COL CGAACGACTGCTTCTCTGAT-3') and W3-CO-R (5'- (ADU05552.1), Phalaenopsis hybrida COL GCCTGTTCTTCCTCTTCTCC-3') primers. Data (ACS94258.1), Cymbidium sinense COL analysis was performed according to RealPlex software (ADN97077.1), C. goeringii COL (ADQ27228.1), (Eppendorf). The PCR reaction was pre-treated with C. ensifolium COL (ADQ27229.1), Erycina pusilla 50°C 2 min followed by initial denaturation for 10 min COL5 (AGI62029.1), Boehmeria nivea COL2 at 95°C, denaturation for 15 s at 95°C, annealing for (AHI45076.1), Solanum lycopersicum COL 30 s at 58°C, and a last extension for 30 s at 72°C. Rela- (AAS67376.1), Arabidopsis thaliana CO/COL1-5 tive expression was calculated by the ΔΔCT method. (NP_197088.1, NP_186887.1, NP180052.1, For photoperiod response analysis, Total RNA was NP197875.2, NP_568863.1, NP_197088.1), Oryza extracted from the leaves of plants treated with long sativa Hd1 (ABB17664.1), Zea mays Hd1 day, neutral day, and short day conditions at 4 h inter- (ABW82153.1), and Triticum aestivum Hd1 vals across 48 h, starting from 0 h. The RNA extraction (BAC92733.1) were aligned using MAFFT (Katoh and and quantitative real-time RT-PCR were performed as Standley, 2013). Protein motifs of DcCOL and other described above. Four replications were performed for COL were predicted using MEME (Bailey et al., 2015). each time point. Results and Discussion Phylogenetic analysis MAFFT-Aligned amino acid sequences of DcCOL DcCOL protein sequence analysis and CO/COL from other plant species were used to find First strand DNA derived from Dendrobium the best model for constructing a phylogenetic tree curmentaum (KT894671) was 969 bp, and encoded 322 using the model test tool in MEGA6 (Tamura et al., amino acids with a molecular weight of 34.25 kDa. The 2013). A phylogenetic tree of these CO/COL proteins deduced DcCOL amino acid sequence shared a high was then constructed using Neighbor-Joining (NJ) identity with COL from Dendrobium (92%), method with a JTT + G model using MEGA6 (Tamura Cymbidium (86%), Phalaenopsis COL (85%), Oryza et al., 2013). Bootstrap values were derived from 1000 sativa Hd1 (70%), Erycina COL5 (66%), Boehmeria replicates. nivea COL2 (44%), Solanum lycopersicum COL (41%), and Arabidopsis thaliana CO (39%). This result dem- Gene expression analysis onstrated that DcCOL has the highest homology with Total RNA was extracted from roots, pseudobulbs, the closely related species, D. lodigisii, and more simi- and leaves of juvenile and reproductive plants of lar with monocot than dicot confirming that DcCOL D. crumenatum. In addition, total RNA was also ex- was one of the COL members in the Dendrobium tracted from inflorescence, floral buds, and flowers of orchid. In addition, according to amino acid sequence D. crumenatum in the reproductive stage. All alignment, three conserved regions were detected in the D. crumenatum plants used in this experiment were cul- DcCOL sequence. The first region, B-box1, comprised tured under the greenhouse conditions mentioned previ- approximately 40 amino acids, starting from C20 to ously. Three plants were used for each stage of H59. The second region, B-box2 comprised 38 amino development. The RNA extraction was performed as acids starting from C65 to H102. The last region, CCT, mentioned previously. Total RNA was reverse tran- which was the highest conserved region comprised 43 scribed into cDNA using a RevertAid First Strand amino acids starting from R246 to R288 (Fig. 1). cDNA Synthesis kit (Thermo Scientific, USA). The MEME suite was used to perform motif analysis to de- first strand synthesis reaction included 1 μg of total lineate the functional motifs in DcCOL protein. The RNA, 5x reaction buffer, 10 mM dNTP Mix, RiboLock predicted motifs of DcCOL included the known CCT RNase Inhibitor 20 U, and RevertAid M-MuLV RT (motif 1) and the B-box domains (motif 2 and motif 3; 200 U. The reaction was incubated for 60 min at 42°C Fig. 2). and terminated at 70°C for 5 min. Quantitative real-time According to the presence and arrangement of puta- RT-PCR was conducted on an Eppendorf Mastercycler® tive motifs, DcCOL was classified into group I B-box ep realplex4 S real-time RT-PCR instrument zinc finger protein. The consensus sequence detected in

(Eppendorf, USA) with Luminaris Color HiGreen DcCOL B-box1 was C-X2-C-X10-C-X7-C-X2-C-X4-H- qPCR Master Mix (Thermo scientific). The quantitative X8-H and B-box2 was C-X2-C-X8-C-X7-C-X2-C-X4-H- real-time RT-PCR reaction included cDNA template X8-H (Fig. 1). There were seven conserved residues, 3 μL, 0.3 μM of forward/reverse primers, Maxima including cysteine and histidine that acted as one zinc SYBR green qPCR Master Mix(2x) no ROX 12.5 μL, atom binding per one B-box in DcCOL protein struc- and water was added up to 25 μL. Ubiquitin was ampli- ture. The B-box domains have been demonstrated to be fied as an internal control using DOUbi-F (5'-AGGC involved in the protein-protein interaction (Torok and TAAGAGGTGGAACAATGATC-3') and DOUbi-R (5'- Etkin, 2001). The consensus sequence of B-box1 and 256 W. Kaewphalug, P. Srifah Huehne and A. Sriboonlert

Fig. 1. Multiple sequence alignment of DcCOL. Multiple sequence alignment of DcCOL and COL from other plant species was performed with MAFFT (Katoh and Standley, 2013). Conserved B-boxes, VP motif, and CCT domain are shown in highlighted areas. The nuclear localization signal in the CCT domain is shown in a box.

B-box2 in Arabidopsis and rice and EpCOL5 are as fol- Cymbidium COLs but were slightly different from lows: Arabidopsis; B-box1: C-X2-C-X7-8-C-X2-D-X-A- EpCOL5. The B-box1 of EpCOL5 contained five addi- X-L-C-X2-C-D-X3-H, B-box2: C-X2-C-X3-P-X4-C-X2- tional amino acids in addition to DcCOL (Fig. 1). Most D-X3-L-C-X2-C-D-X3-H, Rice B-box1: C-X2-C-X8-C- of the CO/COL that contained both B-box1 and B-box2 X7-C-X2-C-X4-H-X8-H, B-box2: C-X2-C-X8-C-X7-C- domains were reported to be involved in photoperiodic X2-C-X4-H-X8-H, EpCOL5; B-box1: C-X2-C-X13-C- control of flowering e.g. Arabidopsis CO (Valverde, X7-C-X2-C-X4-H-X8-H, B-box2: C-X2-C-X8-C-X7-C- 2011), Oryza sativa Hd1 (Yano et al., 2000), Lolium X2-C-X4-H-X8-H. Interestingly, the DcCOL B-box perenne LpCO (Martin et al., 2004), and Hordeum consensuses were identical to other Dendrobium and vulgare HvCO1 (Campoli et al., 2012). However, the Hort. J. 86 (2): 252–262. 2017. 257

Fig. 2. Putative motifs in various COL proteins as identified by MEME. The boxes represent different putative motifs. The expected values for all motifs were calculated by MEME (Bailey et al., 2015). Black, white, and gray boxes represent CCT, B-box I, and B-box II motifs, respectively. Group I CO/COL contains all three motifs, CCT, B-box I, and B-box II motifs. Group II contains a CCT domain, one conserved B- box I and one highly variable B-box II. Group III contains CCT and B-box I motifs.

CO/COL proteins that contain only B-box1 were dem- protein-protein interaction (Robson et al., 2001). The onstrated to be involved in other pathways other than nuclear localization signals (NLSs) can be classified the flowering control (Gangappa and Botto, 2014). into two groups, monopartite and bipartite. The mono- Therefore, the functional B-box2 domain has been sug- partite NLSs contain K-(K/R)-X-(K/R) as a putative gested to be crucial for flower induction (Campoli consensus sequence (Crocco and Botto, 2013; Lange et al., 2012; Griffiths et al., 2003; Liu et al., 2015; et al., 2007). The bipartite NLSs contain a putative con- Martin et al., 2004). Although both the AtCO and sensus sequence of (K/R)-(K/R)-X10-12-(K/R) (Crocco OsHD1 contain both B-box1 and B-box2 domains, the and Botto, 2013). The NLSs identified in the DcCOL, composition of amino acids in these two domains are and other members of CO/COL analyzed was bipartite slightly different. Both of these proteins have a function (Fig. 1). The previous study showed that bipartite NLSs in induction of flowering, but their regulation pathways has a nuclear uptake function in CO and COL3 (Crocco are different. Hd1 was demonstrated to promote flower- and Botto, 2013; Datta et al., 2006; Robson et al., ing in short day (SD) (Hayama et al., 2003; Kim et al., 2001). This segment of the CCT domain was demon- 2008). While AtCO promotes flowering in long day strated to localize GFP to the nucleus. This CCT do- (LD) (Putterill et al., 1995; Samach et al., 2000; Zhang main was demonstrated to have functions in both et al., 2011). The different regulation patterns suggested transcriptional regulation and nuclear localization. A that other parts of this protein are responsible for the mutation analysis of the CCT domain delays flowering regulation of flowering. Another important domain in without affecting the nuclear localization function (Kim the DcCOL was the CCT domain. Most of the CCT et al., 2013; Robson et al., 2001). Apart from these domain-containing proteins have functions associated three domains in the CO/COL, other domains were also with environmental responses to light, temperature, or identified in the CO/COL proteins. The VP Motif the circadian clock (Wenkel et al., 2006). The CCT do- (Fig. 1) located between the B-box2 and CCT was iden- main was demonstrated to have two crucial functions in tified in the structural group I (Crocco and Botto, 2013; the CO/COL proteins; a nuclear localization and a Gangappa and Botto, 2014). This motif has an upstream 258 W. Kaewphalug, P. Srifah Huehne and A. Sriboonlert stretch of 4–5 negatively charged residues and the core prised of group II CO/COL containing two B-boxes, sequence V-P-E/D-φ-G (φ = hydrophobic residue) one highly conserved and one highly variable B-box. (Holm et al., 2001). It was demonstrated to interact with This group II CO/COL was comprised of AtCOL9, 10, CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1), 11, 12, 13, 14, 15, EpCOL3, 4, 6, 8, and 10. The topolo- an E3 ubiquitin ligase that represses photomorphogene- gy and classification of these CO/COL proteins were sis in the dark (Datta et al., 2006). Even though all similar to the trees demonstrated in previous studies group I CO/COL members contain these four conserved (Chou et al., 2013; Zhang et al., 2011) supporting the motifs, their functions are diverse across all members, classification of DcCOL in the group I COL. However, suggesting that the position responsible for the diversi- even though all group I CO/COL proteins contain simi- fied function of CO/COL is unlikely to be found in lar B-boxes and CCT domain structures, they were these conserved regions. demonstrated to have various functions (Gangappa and Botto, 2014; Liu et al., 2015). AtCO and AtCOL5 were Phylogenetic analysis of CO/COL demonstrated to induce flowering in Arabidopsis To examine the relationship among CO/COL genes, (Hassidim et al., 2009; Suarez-Lopez et al., 2001); how- amino acid sequences of 37 CO/COL from 13 plant ever, the other Arabidopsis group I CO/COL, AtCOL1, species were used to construct a phylogenetic tree. The and AtCOL2 were demonstrated to have little effect on phylogenetic tree of these CO/COL sequences can be flowering (Ledger et al., 2001). Nonetheless, the circa- grouped into three clades based on their structural do- dian clock was shown to have an effect on the expres- mains (Fig. 3). Clade I (73 BP) comprised of CO/COL sion of these two genes (Ledger et al., 2001). Although was categorized in group I B-box as most of the pro- not all group I CO/COLs have a direct effect on flower- teins in this clade contain two highly conserved B- ing, all were regulated by light (Gangappa and Botto, boxes, except for EpCOL11, that contained only one 2014). B‑box. This clade was comprised of DcCOL, AtCO, OsHd1, ZmHd1 and TsHd1, AtCOL1, 2, 3, 4, 5, DcCOL expression analysis BnCOL2, BvCOL2, PhalCOL1, 2, EpCOL5, 11, and all DcCOL expression was examined in various tissue other orchid COLs. Clade II (100 BP) comprised of types and under different light conditions to assess the group III CO/COL containing one B-box and a CCT function of DcCOL in flowering. Quantitative real-time domain. This group III CO/COL was comprised of RT-PCR demonstrated that the DcCOL was expressed AtCOL6, 7, 8, EpCOL7 and 12. Clade III (99 BP) com- in all organs in both juvenile and reproductive stages

Fig. 3. Phylogenetic tree of CO/COL proteins from different plant species. The phylogenetic tree was generated by Neighbor-Joining (NJ) using MEGA6.0 (Tamura et al., 2013) with a bootstrap of 1000. The CO/COL were separated into three groups according to their structural domains. Group I contains CCT, B-box I, and B-box II domains. Group II contains a CCT domain, B-box I, and a diverged B-box II. Group III contains CCT and a B-box I domain. DcCOL was classified into the first group. Numbers above branches indicate bootstrap values. Hort. J. 86 (2): 252–262. 2017. 259

(Fig. 4). In the juvenile stage, expression of DcCOL depending on the habit of the plants. The expression of was detected at the highest level in pseudobulbs and the DcCOL in the juvenile phase leaves was lower than in lowest in the leaves. In the reproductive phase, the the reproductive stages, suggesting that DcCOL is re- highest expression was detected in the open flowers and quired in the reproductive stage possibly for floral in- the lowest in the pseudobulbs. The expression of duction through the photoperiod pathway that occurred DcCOL was approximately two fold higher in the re- in the leaves. productive stage than in the juvenile stage, and the To assess whether DcCOL expression is controlled highest level was found in open flowers. This vegeta- by the photoperiod pathway, the expression patterns of tive stage expression of COL was also demonstrated in DcCOL under different light conditions were investi- E. pusilla (Chou et al., 2013) and P. hybrida (Zhang gated. Quantitative real-time RT-PCR of DcCOL was et al., 2011). The expression of EpCOL and PhalCOL carried out in the mature D. crumenatum specimens genes was detected in different tissues including floral under LD, neutral day (ND), and SD conditions. The buds, opened flowers, peduncles, capsules, leaves, and expression levels of DcCOL under LD and ND were roots (Chou et al., 2013; Zhang et al., 2011). The higher than under SD. The DcCOL mRNA expression CO/COL genes were reported to be expressed through- was observed at four hour intervals across 48 h (Fig. 5). out the life cycle of the plant from embryonic develop- The expression levels of DcCOL under all conditions ment through to the reproductive phase in almost all were decreased at the light phase and increased gradual- tissue types (Tiwari et al., 2010). CO/Hd1 proteins in ly to the peak at 16 Zeitgeber time (ZT). This could Arabidopsis and rice were demonstrated to be ex- partly be a result of the pretreatment with the ND con- pressed in the leaves which is a key location of these dition as the expression in all treatments demonstrated proteins to modulate the transcription of FT/Hd3a (Liu the exact same pattern. However, on the second day of et al., 2015; Turck et al., 2008). However, DcCOL ex- the light treatment (24–48 ZT), the expression levels of pression was detected at a higher level in roots and the DcCOL more closely coincided with the day-night pseudobulbs than in the leaves of the juvenile stage cycle. In all treatments, the DcCOL expressions were (Fig. 4) which is not surprising considering the mor- decreased to the basal level at the light phase and phology of this epiphytic orchid, D. crumenatum. This spiked up to the highest level at exactly 4 h after dark- orchid has large pseudobulbs that produce a long stem ness. The expression patterns of DcCOL in all condi- containing 2–10 small leaves. The pseudobulbs and tions were relatively similar, suggesting that day length roots of most epiphytic orchids, including Dendrobium, does not affect flowering in D. crumenatum. This result are the main locations of photosynthesis. The expres- coincides with the flowering behavior of this orchid. sion of PhalCOL corresponded to its morphology as it D. crumenatum is a free flowering species which can processes big green leaves that grow on the stem and flower throughout the year (Holttum, 1949). The diur- does not contain pseudobulbs. The PhalCOL expression nal expression of DcCOL also coincides with the flow- was detected at the highest level in the leaves than in ering habit of D. crumenatum as its flower opens before the roots and stems during the entire vegetative devel- dawn and lasts for only 24 h (Yap et al., 2008). A simi- opment (Zhang et al., 2011). This evidence suggested lar diurnal expression pattern of DcCOL was also ob- that COL can be expressed differently among species served in OsHd1, and BnCOL2 in which both have a

Fig. 4. Expression of DcCOL in different organs at vegetative and reproductive stages. Quantitative real-time RT-PCR (A) of DcCOL was performed using Ubiquitin (DcUbi) as an internal control gene. Juvenile (B) and reproductive (C) stages of D. crumenatum can be distinguished by the presence of an inflorescence stem (arrow) that appeared only in the adult plants. Error bars represent standard error (n = 3). Jr; Juvenile root, Jp; Juvenile pseudobulb, Jl; Juvenile leaf, Rr; Reproductive root, Rp; Reproductive pseudobulb, Rl; Reproductive leaf, Inf; Inflorescence, Fb; Flower bud, Flo; Open flower. 260 W. Kaewphalug, P. Srifah Huehne and A. Sriboonlert

are formed and the anther is almost fully developed and then undergo dormancy. 4) Upon cold induction, these dormant flower buds open (Arditti, 1979). Floral transition in most flowering plants involves two floral enabling (endogenous and hormonal) and two floral promoting pathways (vernalization and photoperiod). The first three steps of D. crumenatum flowering control are likely to be controlled by the floral enabling pathway to develop the inflorescence stem. The last step of D. crumenatum flowering control is controlled by the vernalization pathway (Arditti, 1979; Holttum, 1949; Meesawat and Kanchanapoom, 2007). Taken together, the floral induction behavior in the natural habi- tat of D. crumenatum and the expression patterns of the light-responsive gene DcCOL demonstrated in this study, the photoperiod pathway does not seem to have a crucial role in controlling the expression of DcCOL and the floral induction in D. crumenatum. Instead, the expression of DcCOL was controlled by the dark and light period during the day, corresponding to the flowering time which only occurs just before the sun rises and lasts for only one day. This similar flowering behavior was also observed in morning glory, Ipomoea nil (Yamada et al., 2007). A differential subtraction screen- ing of cDNA of I. nil flower from t = 0 (8:00 a.m.) to 4 h (11:00 a.m.) showed the expression of the CO-Like gene was dramatically decreased suggesting involve- ment of COL in the flowering process in this species (Yamada et al., 2007). Not only floral induction, but also flower opening require environmental factors such Fig. 5. Expression of the DcCOL gene under different day length as temperature and circadian rhythm (van Doorn and conditions. Expression of the DcCOL gene under long day, neu- van Meeteren, 2003; van Doorn and Kamdee, 2014). tral day, and short day conditions were compared. The expression level of DcCOL in D. crumenatum was determined by Therefore, COL, which is already known for its func- quantitative real-time RT-PCR using specific primers. Day and tion in floral transition, may also have an indirect func- night periods are labeled at the bottom of each chart using tion in flower opening as demonstrated in this study. white and black bars, respectively. Transcripts of DcCOL were Another factor that controlled flowering in determined using four replicates and normalized with D. crumenatum was demonstrated to be induction by Ubiquitin. Experimental time refers to Zeitgeber time (ZT). Error bars represent standard error (n = 4). vernalization. Cold induction was detected in various tropical epiphytic orchid species e.g. Phalaenopsis aphrodite (Jang et al., 2015), Dendrobium Second Love function in flowering control (Izawa et al., 2002; Liu (Campos and Kerbauy, 2004), and D. nobile (Yen et al., et al., 2015). Taken together, the results from protein 2008). In Phalaenopsis aphrodite, diurnal fluctuation of structure analysis, phylogenetic and expression patterns high day and low night temperatures or a cool tempera- suggested that DcCOL can be classified in group I CO ture at night promoted flowering of this orchid. A low similar to Arabidopsis CO, rice Hd1, and BnCOL2 that temperature, but not photoperiod, was demonstrated to are all suggested to have a function involved in flower- induce flowering by directing the expression of the two ing regulation. Moreover, expression analysis of main floral integrator genes, FT and FD (Jang et al., DcCOL demonstrated a diurnal expression pattern and 2015). accumulation of transcript during the dark similar to the expression pattern in OsHd1 and BnCOL2, suggesting Conclusions that DcCOL may also have a function in flowering con- This study demonstrated that the expression of trol in the orchid D. crumenatum. DcCOL, the main photoperiodic responsive gene in The flowering control in D. crumenatum is a com- D. crumenatum, was not affected by the photoperiod, plex process involving the following steps: 1) An in- but its expression responded to light and dark periods in duction of inflorescence development on the terminal a daily cycle. Although the direct effect of DcCOL ex- portion of stems. 2) Development of flower buds on the pression and floral induction was not demonstrated in stem. 3) The flower buds develop until all flower parts this study, the expression patterns reported here coin- Hort. J. 86 (2): 252–262. 2017. 261 cide with the flowering behavior of this orchid in the 4688. natural environment, suggesting the function of DcCOL Fu, C.-H., Y.-W. Chen, Y.-Y. Hsiao, Z.-J. Pan, Z.-J. Liu, Y.-M. in flowering control. Huang, W.-C. Tsai and H.-H. Chen. 2011. OrchidBase: a collection of sequences of the transcriptome derived from Acknowledgements orchids. Plant Cell Physiol. 52: 238–243. 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