Plant Cell Physiol-2013-Ding-595-608 (1)

Overexpression of DOSOC1,an Ortholog of Arabidopsis SOC1,Promotes Flowering in the Orchid Dendrobium Chao Parya Smile

Lihua Ding1,Yanwen Wang1and Hao Yu1,2,*

1Department of Biological Sciences,Faculty of Science,National University of Singapore,Singapore117543,Singapore

2Temasek Life Sciences Laboratory,National University of Singapore,1Research Link,Singapore117604,Singapore

*Corresponding author:E-mail,dbsyuhao@https://www.wendangku.net/doc/559962913.html,.sg;Fax,+65-67792486.

(Received November20,2012;Accepted February1,2013)

SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1(SOC1) encodes a MADS-box protein that plays an essential role in integrating multiple?owering signals to regulate the transi-tion from vegetative to reproductive development in the model plant Arabidopsis.Although SOC1-like genes have been isolated in various angiosperms,its orthologs in Orchidaceae,one of the largest families of?owering plants, are so far unknown.To investigate the regulatory mechanisms of?owering time control in orchids,we isolated a SOC1-like gene,DOSOC1,from Dendrobium Chao Praya Smile.DOSOC1 was highly expressed in reproductive organs,including in?or-escence apices,pedicels,?oral buds and open?owers.Its expression signi?cantly increased in whole plantlets dur-ing the transition from vegetative to reproductive develop-ment,which usually occurred after8weeks of culture in Dendrobium Chao Praya Smile.In the shoot apex at the ?oral transitional stage,DOSOC1was particularly expressed in emerging?oral meristems.Overexpression of DOSOC1in wild-type Arabidopsis plants resulted in early?owering, which was coupled with the up-regulation of two other?ow-ering promoters,AGAMOUS-LIKE24and LEAFY.In addition, overexpression of DOSOC1was able partially to complement the late-?owering phenotype of Arabidopsis soc1-2loss-of-function mutants.Furthermore,we successfully created seven35S:DOSOC1transgenic Dendrobium orchid lines, which consistently exhibited earlier?owering than wild-type orchids.Our results suggest that SOC1-like genes play an evolutionarily conserved role in promoting?owering in the Orchidaceae family,and that DOSOC1isolated from Dendrobium Chao Praya Smile could serve as an import-ant target for genetic manipulation of?owering time in orchids.

Keywords:Arabidopsis Flowering time MADS-box gene Orchid SOC1.

Abbreviations:BA,benzyladenine;bar,bialaphos resistance; CaMV,Cauli?ower mosaic virus;MSO,L-methionine sulfoxi-mine;PLB,protocorm-like body;RACE,rapid ampli?cation of cDNA ends;RT–PCR,reverse transcription–PCR.The nucleotide sequence reported in this paper has been sub-mitted to GenBank under the accession number:DOSOC1, KC121576.

Introduction

The transition from vegetative to reproductive phase,known as the?oral transition,is one of the most dramatic developmental switches in the life cycle of a?owering plant.Determining the optimal timing for the?oral transition is critical for?owering plants to achieve reproductive success.Over the past three decades,intensive molecular genetic analyses using the model plant Arabidopsis thaliana have revealed a complex genetic network in the control of this transition.This network consists of multiple?owering genetic pathways,which respond to vari-ous endogenous and environmental signals(Mouradov et al. 2002,Simpson and Dean2002,Boss et al.2004).The inter-actions among these?owering pathways regulate the expres-sion of several?oral pathway integrators,including SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1(SOC1), which in turn mediate other regulators,such as the?oral meri-stem identity gene LEAFY(LFY),to determine the formation of ?oral meristems(Borner et al.2000,Lee et al.2000,Samach et al. 2000,Moon et al.2003,Liu et al.2008,Wang et al.2009).

SOC1encodes a MADS-box protein,a member of a large family of transcription factors in plants that share a highly conserved DNA-binding motif,the MADS-box domain.SOC1 mRNA is expressed in leaves and shoot apical meristems,and its expression levels are tightly controlled by multiple?owering pathways(Borner et al.2000,Lee et al.2000,Samach et al. 2000,Moon et al.2003,Wang et al.2009,Shen et al.2011).A protein complex consisting of two?owering repressors, FLOWERING LOCUS C(FLC)and SHORT VEGETATIVE PHASE(SVP),suppresses SOC1expression at the vegetative phase,while several?owering pathways,including autono-mous,gibberellin and vernalization pathways,down-regulate FLC and SVP,thus derepressing SOC1during the?oral transition (Helliwell et al.2006,Searle et al.2006,Li et al.2008).In the

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photoperiod pathway,CONSTANS (CO ),which plays a central role in mediating photoperiod-dependent ?owering (Putterill et al.1995),activates SOC1expression either directly or indir-ectly via another ?oral pathway integrator,FLOWERING LOCUS T (FT)(Samach et al.2000,Hepworth et al.2002,Wigge et al.2005,Yoo et al.2005).In the age-dependent path-way,miRNA156-targeted SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL)transcription factors up-regulate SOC1(Wang et al.2009),while the gibberellin pathway particularly promotes SOC1expression under non-inductive short-day con-ditions (Moon et al.2003).

SOC1and another ?oral pathway integrator AGAMOUS-LIKE 24(AGL24)directly regulate mutual mRNA expression at the shoot apex during the ?oral transition (Liu et al.2008).Upon its interaction with AGL24,SOC1protein is translocated into the nucleus to act as a transcription factor (Lee et al.2008).In addition,the conformational dynamics of these two proteins regulated by a PIN1-type parvulin 1,Pin1At,affects their pro-tein stability in the nucleus (Wang et al.2010).Furthermore,two recent studies on SOC1DNA-binding pro?les using ChIP-chip and ChIP-seq have revealed that SOC1binds to its own promoter to activate transcription (Immink et al.2012,Tao et al.2012).These observations suggest that SOC1and AGL24function together to mediate the integration of ?ower-ing signals perceived by them,which provides swift positive feedback regulation of their mRNA expression required for the synergistic promotion of ?owering when environmental and developmental conditions are permissible for reproductive development.

SOC1belongs to the Tomato MADS-box gene 3(TM3)clade of MADS-box genes,which comprises genes from dicots,mono-cots and gymnosperms,indicating that SOC1-like genes may contribute to some ancestral functions relevant to vegetative development in addition to their known functions in ?owering and ?ower development (Lee et al.2000,Becker and Theissen 2003,Cseke et al.2003,Ferrario et al.2004,Lee et al.2004,Nakamura et al.2005).Although SOC1-like genes have been isolated in a wide range of plant species (Lee and Lee 2010),so far the accumulating research data mainly concern their expression patterns and sequence information.Therefore,fur-ther functional characterization of SOC1-like genes in various plants is required for understanding their functional conserva-tion and divergence during evolution.

Orchids are members of the family Orchidaceae,which is one of the largest families of ?owering plants.While orchids are major ornamental plants as popular cut ?owers or potted plants throughout the world,traditional breeding methods of sexual hybridization and selection are too time-consuming to meet the increasing demand for rapid production of commer-cially valuable orchids.One major obstacle for orchid breeding is the prolonged vegetative phase before orchids switch to re-productive development (Yu and Goh 2001).Thus,elucidation of the molecular mechanisms of the ?oral transition in orchids is important for identifying key regulators required for targeted genetic manipulation of ?owering traits in orchids.Although

several attempts have been made to clarify orchid genes involved in the ?oral transition (Yu and Goh 2000a,Yu and Goh 2000b,Yu et al.2000,Hsu et al.2003,Hou and Yang 2009),SOC1-like genes and their effects on ?owering in orchids are hitherto unknown.In this study we report the isolation and functional analysis of a SOC1ortholog,DOSOC1,from a Dendrobium orchid.Sequence and expression analysis,and transgenic studies in both Arabidopsis and orchids suggest that DOSOC1plays an evolutionarily conserved role in the pro-motion of orchid ?owering.

Results

Isolation of DOSOC1from Dendrobium Chao Praya Smile

To facilitate molecular genetic studies in orchids,we have pre-viously created an ef?cient and reproducible gene transform-ation system utilizing L -methionine sulfoximine (MSO)as an agent for selection of transgenic Dendrobium Chao Praya Smile with the bialaphos resistance (bar )gene as a selectable marker (Chai et al.2007).In addition,in vitro orchid culture systems have been developed for several Dendrobium hybrids,including Dendrobium Chao Praya Smile (Yu and Goh 2000a,Hee et al.2007,Sim et al.2007).These efforts have established Dendrobium Chao Praya Smile as a tractable system for inves-tigating key regulatory genes in the control of orchid development.

To isolate SOC1-like genes from Dendrobium Chao Praya Smile,we compared the protein sequences of SOC1-like genes from various ?owering plants,and designed degenerate forward and reverse primers from the conserved regions of the K domain and the SOC1motif at the C-terminus (Vandenbussche et al.2003,Nakamura et al.2005),respectively (Fig.1A ).A cDNA fragment of around 300bp was ampli?ed by reverse transcription–PCR (RT–PCR)using total RNA extracted from leaves.As sequence alignment revealed high similarity of this fragment to SOC1orthologs from various plant species,we further obtained the full-length cDNA sequence,designated DOSOC1(GenBank accession No.KC121576),using the rapid ampli?cation of cDNA ends (RACE)method.

DOSOC1cDNA is 725bp in length with a 669bp coding region.Similar to other known MADS-box genes,the deduced amino acid sequences of DOSOC1contain a well-conserved MADS domain,a less conserved K domain,and a divergent C-terminal region (Fig.1A ).DOSOC1shared 52%sequence identity with Arabidopsis SOC1,and shared even higher se-quence identity with SOC1orthologs in other monocots,such as EgAGL20(Elaeis guineensis ;63%identity),OsSOC1(Oryza sativa ;57%identity)and ZmMADS1(Zea mays ;56%identity).Multiple sequence alignment of DOSOC1and other SOC1-like proteins further con?rmed the presence of a well-conserved SOC1motif containing 11amino acid residues at their C-termini (Fig.1A ),which is only speci?c in the TM3clade of MADS-box genes from gymnosperms and angiosperms

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(Vandenbussche et al.2003,Nakamura et al.2005).These se-quence analyses indicate that DOSOC1might be a SOC1ortho-log in the orchid Dendrobium Chao Praya Smile.Interestingly,three amino acid residues,valine (V;second),leucine (L;sixth)and glycine (G;ninth),in the SOC1motif are identical in all SOC1-like proteins examined (Fig.1A ).As these three amino acid residues all belong to the aliphatic group of amino acids,whether their similar structure and general

chemical

Fig.1Analysis of the DOSOC1sequence.(A)Alignment of the amino acid sequences of DOSOC1and its orthologs from other plant species.Black and gray boxes indicate identical and similar residues,respectively.The regions of the MADS domain,K domain and the speci?c SOC1motif are underlined.Arrows indicate the conserved amino acid sequences that were used for designing degenerate forward and reverse primers to amplify SOC1-like genes in Dendrobium Chao Praya Smile.The protein sequences of SOC1-like genes aligned in this study were retrieved from NCBI.The names of respective species are given behind the corresponding protein names as follows:IbAGL20,Ipomoea batatas ;CmSOC1,Chrysanthemum ;FvSOC1,Fragaria vesca ;SOC1,Arabidopsis thaliana ;BrAGL20,Brassica rapa ;GmSOC1,Glycine max ;PsSOC1a,Pisum sativum ;VvMADS8,Vitis vinifera ;PtSOC1,Populus trichocarpa ;MvSOC1,Magnolia virginiana ;EgAGL20-like,Elaeis guineensis ;DOSOC1,Dendrobium Chao Praya Smile;OsSOC1,Oryza sativa ;ZmMADS1,Zea mays ;TaAGL20,Triticum aestivum .A non-SOC1-like protein,DEFICIENS-AGAMOUS-LIKE2(DAL2)(X79280)of Picea abies ,was included as an outlier in the sequence alignment.(B)Southern blot analysis of the DOSOC1gene.The DNA gel blot containing 20m g of genomic DNA digested with Eco RV,Bam HI and Spe I was hybridized with the digoxigenin-labeled probe that was synthesized from the C-terminal speci?c region of DOSOC1.The sizes of the DNA markers are given on the right in kilobases.

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characteristics contribute to the speci?c function of SOC1-like proteins in plants remains an intriguing question to be inves-tigated further.

To investigate the genomic organization of DOSOC1,we performed Southern blot analysis of orchid genomic DNA di-gested with Eco RV,Bam HI and Spe I using the digoxigenin-labeled probe from the C-terminal gene-speci?c region of DOSOC1(Fig.1B ).The result indicates that DOSOC1is present either as a single-copy gene or in low copy number in the genome of Dendrobium Chao Praya Smile.

A phylogenetic tree based on the analysis of the MIK region was further constructed to determine the evolutionary rela-tionship between DOSOC1and other SOC1-like proteins,(Fig.2).The tree showed that DOSOC1was clustered in Clade 1that mostly included SOC1and its orthologs from eudicots,such as GmSOC1from Glycine max and BrAGL20from Brassica rapa ,whereas the other SOC1orthologs from monocots,such as OsSOC1from O.sativa and ZmMADS1from Z.mays ,were clustered in Clade 2.

Temporal and spatial expression patterns of DOSOC1

To investigate genes involved in the ?oral transition,we opti-mized an in vitro orchid culture system for Dendrobium Chao Praya Smile (Fig.3)based on the approaches established for several Dendrobium hybrids (Yu and Goh 2000a,Hee et al.2007,Sim et al.2007).This system allowed rapid in vitro development of Dendrobium Chao Praya Smile from the vegetative to repro-ductive phase within about 3months,which overcame the time constraint for investigating ?owering time genes in many other orchids that could have a long juvenile phase up to 30months (Hee et al.2007).Under our culture conditions,the starting materials,which were thin sections of protocorms,generated protocorm-like bodies (PLBs)about 0.5cm in length within 4weeks (Fig.3).These PLBs further developed into vegetative plantlets over the next 4weeks.Most of these plant-lets produced the typical transitional shoot apical meristem as de?ned by a previous study (Yu and Goh 2000a)with narrow-ing of the two youngest visible leaves toward the apex after another 4weeks in culture,after which the plantlets entered into the reproductive stage with normal in?orescence and ?ower development.

To characterize the function of DOSOC1,we ?rst examined the spatial expression of DOSOC1in various orchid tissues by quantitative real-time PCR.DOSOC1transcripts were detect-able at very low levels in vegetative tissues,such as roots,stems and leaves,whereas its expression in reproductive organs,such as in?orescence apices,pedicels,?oral buds and open ?owers,signi?cantly increased (Fig.4A ).These results indicate that DOSOC1function might be closely associated with the repro-ductive development of Dendrobium Chao Praya Smile.

To understand whether DOSOC1plays a role similar to SOC1in regulating ?owering time,we studied the temporal DOSOC1expression in whole plants at various developmental stages of

Dendrobium Chao Praya Smile growing in our in vitro culture system (Fig.4B ).DOSOC1expression was low in 3-week-old PLBs and 5-week-old vegetative plantlets,and dramatically increased in the plantlets progressing from the late vegetative stage to the ?oral transitional stage.Its expression reached the maximal level in 11-week-old plantlets at the transitional stage,and gradually decreased in the plantlets that were developing in?orescences and ?owers.The observation of up-regulation of DOSOC1in Dendrobium Chao Praya Smile is similar to the increased SOC1expression in Arabidopsis during the ?oral tran-sition,implying that DOSOC1may play a conserved role in promoting ?owering in orchids.

We then performed in situ hybridization to examine the localization of DOSOC1transcripts in shoot apices of Dendrobium Chao Praya Smile in order to gain further insight into DOSOC1function.Consistent with its low expression in vegetative plantlets measured by quantitative real-time PCR,DOSOC1was barely detectable in the 6-week-old vegetative shoot apex (Fig.5A ).In a 10-week-old plantlet at the ?oral transitional stage,DOSOC1was speci?cally expressed in an emerging ?oral meristem and the vascular tissues below the shoot apical meristem,but not in the shoot apical meristem itself (Fig.5B ).Thus,DOSOC1exhibits a distinct expression pattern in the in?orescence apex from SOC1in Arabidopsis,which is up-regulated in the in?orescence shoot apical meri-stem during the ?oral transition,but down-regulated in emer-ging ?oral meristems (Lee et al.2000,Liu et al.2007).The distinct expression patterns of DOSOC1and SOC1in the in?or-escence apex indicate the possible divergence of their func-tional modes in orchids and Arabidopsis during the ?oral transition,although both of them are up-regulated during the transition from vegetative to reproductive development.

DOSOC1promotes ?owering in Arabidopsis

We created transgenic Arabidopsis plants in which DOSOC1was driven by the Cauli?ower mosaic virus (CaMV)35S pro-moter to explore the biological function of DOSOC1.Out of 18independent 35S:DOSOC1transgenic Arabidopsis lines ob-tained at the T 1generation,four lines were phenotypically in-distinguishable from wild-type plants that produced 9–11rosette leaves when bolting under long days,while all the other transgenic lines showed earlier ?owering typically with seven and eight rosette leaves (Fig.6A ,B ).Consistent with the early-?owering phenotype observed in 35S:DOSOC1transgenic Arabidopsis lines,DOSOC1was highly expressed in a represen-tative 35S:DOSOC1line,but not in a wild-type Arabidopsis plant (Fig.6A ,C ).

We further overexpressed DOSOC1in Arabidopsis soc1-2loss-of-function mutants to test whether DOSOC1could com-pensate for the loss of SOC1.Twenty independent soc1-235S:DOSOC1transgenic Arabidopsis lines were obtained at the T 1generation (Fig.6A ,B ).Among these lines,four soc1-235S:DOSOC1transgenic lines showed ?owering time similar to soc1-2,which displayed late ?owering with 18–20rosette leaves

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under long days.Another 16soc1-235S:DOSOC1transgenic lines exhibited rescue of late ?owering of soc1-2to different extents.Consistently,DOSOC1was highly expressed in a repre-sentative soc1-235S:DOSOC1line that ?owered earlier than soc1-2,but not in a soc1-2mutant (Fig.6A ,C ).

As SOC1accelerates ?owering through up-regulating other downstream ?owering promoters,such as AGL24and LFY ,in Arabidopsis (Yu et al.2002,Lee et al.2008),we then examined whether these genes were similarly affected by DOSOC1in 35S:DOSOC1transgenic lines.Expression analysis demonstrated that the expression of both AGL24and LFY was evidently ele-vated in 5-day-old 35S:DOSOC1as compared with wild-type seedlings (Fig.7).

These observations suggest that DOSOC1plays a role like that of SOC1in the promotion of ?owering partially through engaging similar downstream regulators in Arabidopsis.However,we noticed that in Arabidopsis,the degree of early ?owering promoted by overexpression of DOSOC1(Fig.6B )was less than what was observed for overexpression of SOC1(Lee et al.2000,Liu et al.2007).In addition,35S:DOSOC1

was

Fig.2Phylogenetic analysis of DOSOC1and its orthologs from various plant species.The phylogenetic tree was generated with the Neighbor–Joining algorithm.DAL2was chosen as an outgroup.Bootstrap values (>50%)in 1,000replicates are indicated next to the

nodes.

Fig.3Main developmental stages during in vitro cultivation of orchid Dendrobium Chao Praya Smile from PLB formation to ?ower develop-ment.Thin sections of protocorms were used as starting materials and cultured in modi?ed liquid KC medium to induce the formation of protocorm-like bodies (PLBs)within 4weeks.PLBs further develop into plantlets at the vegetative and transitional stage in liquid medium in the next 8weeks.After the ?oral transition from vegetative to reproductive growth,plantlets were transferred to two-layer modi?ed KC medium as stated in the Materials and Methods for further in?orescence and ?ower development.The duration of each developmental stage is de?ned according to the similar status of >60%of the plantlets examined.Scale bars,5mm.

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only able to rescue partially the late-?owering phenotype of soc1-2(Fig.6B ).Thus,although DOSOC1and SOC1share the conserved promoting function during the ?oral transition,they could evolve with different molecular features in orchids and Arabidopsis,respectively.

Generation of 35S:DOSOC1transgenic orchids

To investigate further the endogenous function of DOSOC1in orchids,we created transgenic Dendrobium Chao Praya Smile harboring 35S:DOSOC1in a pGreen vector through integrating an orchid gene transformation system utilizing MSO as a selec-tion agent for the bar gene (Chai et al.2007)with the optimized in vitro orchid culture system (Fig.3).

After particle bombardment of thin sections of wild-type calli,the transformed materials were subjected to three differ-ent stages of culture for subsequent selection of putative trans-formants (Fig.8).The bombarded orchid cells recovered from physical damage on the medium without MSO at the ?rst stage of recovery culture.However,as unbombarded wild-type cells

could outcompete bombarded cells on this medium,the ideal length for this stage was 4d for Dendrobium Chao Praya Smile as previously reported (Chai et al.2007).At the second and third stages,the bombarded thin sections were selected on the medium containing 0.5and 2m M MSO for initial and lethal selection,respectively.Surviving calli produced by bombarded tissues were cut into small pieces and subcultured onto fresh medium every 2weeks to avoid generating chimeric transgenic orchids.In this selection system,almost all unbombarded wild-type Dendrobium Chao Praya Smile tissues turned necrotic and eventually died,whereas some putative transformed calli survived and proliferated into PLBs after 3months (Fig.8).Thin sections from these putative transgenic PLBs and their derived PLBs were further cultured in the optimized in vitro culture system (Fig.3)for further characterization of transgenic plants.

DOSOC1promotes ?owering in Dendrobium Chao Praya Smile

We created seven independent 35S:DOSOC1transgenic Dendrobium Chao Praya Smile lines using the above-mentioned MSO selection coupled with an in vitro culture system.The presence of the 35S:DOSOC1transgene in these plants was veri?ed by PCR genotyping using the speci?c primers from 35S and DOSOC1(Fig.9A ).In our in vitro culture system,all of these transgenic lines produced the ?rst visible in?orescence stalks at 12–15weeks of culture (Fig.9B ).In contrast,wild-type orchid plants produced in?orescence stalks at the earliest at 17weeks of culture,and nearly half of the wild-type plants exam-ined did not produce in?orescences within 24weeks (Figs.9B ,10A ).These results demonstrate that overexpression of DOSOC1contributes to early ?owering of Dendrobium Chao Praya Smile.

We measured DOSOC1expression levels in the leaves of two representative transgenic orchids that produced the ?rst visible in?orescence stalks at 14weeks of culture (Fig.10A ).As ex-pected,DOSOC1was highly expressed in these two 35S:DOSOC1lines,whereas its expression in wild-type leaves was relatively low (Fig.10B ).

After the ?oral transition,we further cultured these trans-genic plants in the in vitro culture system and observed their ?oral phenotypes.Among seven transgenic orchid lines,only two lines developed the in?orescence apices,each of which eventually produced at least one normal ?ower (Fig.10C ,left panel),whereas in?orescence and ?ower development of the other lines were all abolished,with the generation of abnormal ?oral buds on the apex,most of which only developed initial perianth organs without reproductive organs (Fig.10C ,right panel).Under the same in vitro culture conditions,nearly half of the ?owering wild-type plantlets could develop normal ?owers as previously reported (Hee et al.2007).Thus,the ratio of generating normal ?owers was much lower in 35S:DOSOC1transgenic orchids than in wild-type orchids,implying that overexpression of DOSOC1compromises normal ?ower

development.

Fig.4Quantitative analysis of DOSOC1expression in various tissues and at different developmental stages of Dendrobium Chao Praya Smile.(A)Expression of DOSOC1in various tissues.Rt,root;Sm,stem;Lf,leaf;IA,in?orescence apex;Pl,pedicel;FB,?oral bud;OF,open ?ower.(B)Time-course of expression of DOSOC1in orchid plants at various developmental stages as described in Fig.3.PLB,protocorm-like bodies;VS,vegetative stage;TS,transitional stage;IFD,in?orescence and ?ower development.Transcript levels in (A)and (B)were determined by quantitative real-time PCR analyses of three independently collected samples.Expression results were nor-malized against the expression of the orchid polyubiqutin gene (DOUbi ).Error bars indicate the SD.

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Discussion

Precise control of ?owering time is an essential developmental process that determines reproductive success of ?owering plants.Although Orchidaceae is one of the largest and most widespread families of ?owering plants,the molecular mechan-isms underlying the ?oral transition in this family are largely unknown.In this study,we report the isolation and functional analysis of a SOC1ortholog,DOSOC1,from Dendrobium Chao Praya Smile.

Molecular characterization of DOSOC1has provided several pieces of evidence supporting that DOSOC1plays an evolution-arily conserved role like that of SOC1in Arabidopsis in the promotion of ?owering in orchids.First,in addition to the conserved MADS domain and K domain,DOSOC1and other SOC1orthologs share another well-conserved speci?c SOC1motif at the C-termini (Vandenbussche et al.2003,Nakamura et al.2005).Secondly,DOSOC1expression greatly increases in the whole plantlets during the transition from vegetative to reproductive development in orchids.This expression pattern is similar to that of SOC1in Arabidopsis (Borner et al.2000,Lee et al.2000).Thirdly,overexpression of DOSOC1in both Arabidopsis and orchids causes early ?owering,showing the effect of DOSOC1on accelerating ?owering as shown by SOC1.Furthermore,overexpression of DOSOC1partially

complements the late-?owering phenotype of Arabidopsis soc1-2loss-of-function https://www.wendangku.net/doc/559962913.html,stly,overexpression of DOSOC1in Arabidopsis up-regulates another two ?owering promoters,AGL24and LFY ,both of which are known to be directly up-regulated by SOC1during the ?oral transition (Yu et al.2002,Lee et al.2008,Liu et al.2008).All these observations suggest the functional similarity between DOSOC1and SOC1in promoting ?owering during the ?oral transition.

So far MADS-box genes in the SOC1/TM3clade have been isolated from a wide range of plant species,including dicots,monocots and gymnosperms.While most of these studies have presented gene expression patterns and sequence information,the concrete evidence relevant to the function of SOC1ortho-logs is still fairly limited.In particular,there are only a few studies that have investigated the effect of change in the ex-pression of SOC1orthologs in their homologous systems.Alteration of the expression of a Brassica SOC1ortholog,BrAGL20,affects ?owering in Brassica campestris like SOC1(Kim et al.2003).Overexpression of OsSOC1/OsMADS50in rice and UNSHAVEN (UNS )in petunia promotes ?owering,and knockdown of OsSOC1/OsMADS50in rice delays ?owering (Ferrario et al.2004,Lee et al.2004).Furthermore,overexpres-sion of SOC1orthologs from both monocots and dicots in the heterologous system,the model plant Arabidopsis,

consistently

Fig.5In situ localization of DOSOC1transcripts in shoot apices of Dendrobium Chao Praya Smile.(A)DOSOC1is not detectable in serial sections of a 6-week-old vegetative shoot apex.Asterisks indicate vegetative shoot apical meristems.(B)DOSOC1is speci?cally expressed in emerging ?oral meristems (arrowheads)and vascular tissues (arrows)in serial sections of a 10-week-old shoot apex at the ?oral transitional stage.Asterisks indicate in?orescence shoot apical meristems.Scale bars in (A)and (B),200m m.

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accelerates ?owering (Shitsukawa et al.2007,Tan and Swain 2007,Nakano et al.2011).These results are in agreement with our observation on the function of DOSOC1in promoting ?ow-ering in orchids,suggesting that SOC1/TM3-like genes share a primary conserved role as ?owering promoters in angiosperms.SOC1-like genes have also been shown to affect ?ower de-velopment in several plant species.After the ?oral transition in Arabidopsis,SOC1expression remains at an appropriate level in emerging ?oral meristems,which is regulated by another ?oral meristem identity gene APETALA1(Liu et al.2007).Up-regulation or down-regulation of SOC1expression in ?oral meristems is partially responsible for the reversion of ?oral meristems into in?orescence meristems or precocious develop-ment of ?oral organs,respectively (Gregis et al.2006,Liu et al.2007,Liu et al.2009).It has been shown that overexpression of Gh-SOC1in gerbera disturbs ?oral organ development (Ruokolainen et al.2011),and that overexpression of UNS in petunia results in the unshaven ?oral phenotype with ectopic trichome formation on ?oral organs and conversion of petals into leaf-like organs (Ferrario et al.2004).Similarly,we have demonstrated that overexpression of DOSOC1abolishes normal ?ower development with only generation of undevel-oped perianth organs in orchids.These observations indicate that SOC1-like genes also mediate the subtle development of ?oral organs.

In addition,SOC1-like genes may share another function in controlling the life cycle of annual and perennial plants.In Arabidopsis,SOC1and another ?owering time

gene,

Fig.6Overexpression of DOSOC1in Arabidopsis causes early ?owering.(A)35S:DOSOC1in both wild-type and soc1-2genetic backgrounds accelerates ?owering.A wild-type Arabidopsis plant shows later ?owering than a representative 35S:DOSOC1plant at 28d after germination under long days (left panel),while a soc1-2mutant shows later ?owering than a representative soc1-235S:DOSOC1plant at 35d after germination under long days (right panel).(B)Distribution of ?owering time in T 1transgenic plants carrying 35S:DOSOC1in wild-type and soc1-2backgrounds under long days.(C and D)Examination of DOSOC1expression in representative 35S:DOSOC1(C)and soc1-235S:DOSOC1(D)plants shown in (A)by semi-quantitative PCR.The Arabidopsis TUB2gene was ampli?ed as a control.

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FRUITFULL (FUL ),are expressed in in?orescence shoot apices and procambial strands of developing in?orescences,and pro-mote the determinacy of shoot meristems (Hempel et al.1997,Borner et al.2000,Lee et al.2000,Melzer et al.2008).Down-regulation of both genes results in secondary growth and longevity of Arabidopsis plants,which mimics the lifestyle of perennial plants.Interestingly,both a poplar SOC1ortholog,Populus tremuloides MADS-box 5(PTM5),and DOSOC1shown in this study are expressed in vascular tissues,implying that SOC1-like genes could similarly affect the life cycle of many ?owering plants.In orchids,the phenotype of reversion of in-?orescence meristems into vegetative meristems,which is char-acteristic of the secondary growth shown in soc1ful double mutants in Arabidopsis,has been reported in some monopodial orchids (Goh 1976).Thus,it would be interesting to investigate whether orchid SOC1orthologs are also involved in determin-ing the life cycle of orchids.

While SOC1-like genes share similar functions as discussed above,there is also evidence indicating their functional diver-gence.For example,distinct expression patterns of SOC1-like genes in various plants imply that they may be differentially regulated to contribute to the complex regulatory network of the ?oral transition.SOC1,OsSOC1/OsMADS50and DOSOC1all play a role in accelerating ?owering in Arabidopsis,rice and

orchid,respectively,but display distinct expression patterns during the ?oral transition.SOC1is speci?cally up-regulated in the in?orescence shoot apical meristem,but down-regulated in emerging ?oral meristems (Borner et al.2000,Lee et al.2000,Liu et al.2007).This expression pattern is consistent with the role of SOC1in integrating multiple ?owering signals and mediating the processes of ?oral meristem development and ?oral patterning.However,in rice,OsSOC1/OsMADS50is barely detectable in the in?orescence apical meristem and emerging ?oral meristems,but highly expressed in

leaves

Fig.8Genetic transformation of Dendrobium Chao Praya Smile using the MSO selection system.Thin sections of wild-type calli induced from protocorms were placed on solid media on a Petri dish (top left panel),and transformed by particle bombardment with a pGreen vector,in which DOSOC1is driven by two CaMV 35S promoters.After bombardment,thin sections were maintained for recovery cul-ture on solid medium without the selection agent,MSO,for 4d (top right panel).Thin sections were then transferred to solid medium containing 0.5m M MSO for initial selection of putative transformants (middle panels).Surviving calli were cut into small pieces and sub-cultured onto fresh medium every 2weeks.After 6weeks,surviving calli were transferred to lethal selection medium containing 2m M MSO and also subcultured onto fresh medium every 2weeks (bottom panels).After 6weeks,proliferated PLBs from surviving calli were further cultured on lethal selection medium before they were used for further

studies.

Fig.7Overexpression of DOSOC1in Arabidopsis upregulates AGL24and LFY.Transcript levels of AGL24and LFY were determined by quantitative real-time PCR analyses of 5-day-old wild-type and 35S:DOSOC1seedlings.Expression results were normalized against the TUB2expression.Error bars indicate the SD.

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(Komiya et al.2009).This expression pattern indicates that the rice SOC1ortholog could regulate other ?owering regulators in leaves,but may not be directly involved in ?oral meristem de-velopment.In this study,we have shown that DOSOC1exhibits another unique expression pattern,which has not been found for any other SOC1ortholog.DOSOC1is very speci?cally ex-pressed in emerging ?oral meristems,but not in the in?ores-cence shoot apical meristem (Fig.5B ).Given the ?oral defects observed in 35S:DOSOC1transgenic orchids,it is reasonable to hypothesize that DOSOC1function could be closely relevant to ?oral meristem development.

In summary,we have investigated the ?rst SOC1ortholog,DOSOC1,in the Orchidaceae family using a unique MSO selection-based gene transformation coupled with in vitro cul-ture system.Molecular characterization of this gene has not only revealed the functional conservation and divergence of SOC1orthologs,but has also shed new light on the mechanisms underlying the ?oral transition in orchids.As the vegetative phase in orchids is usually a lengthy process that signi?cantly affects the ef?ciency of orchid breeding,identi?cation of key ?owering regulators such as DOSOC1will contribute to improv-ing orchid ?owering traits through either classical breeding or novel genetic engineering approaches.

Materials and Methods

Plant materials and growth conditions

Dendrobium Chao Praya Smile,a hybrid of Dendrobium Pinky and Dendrobium Kiyomi Beauty,were used in this study.In our in vitro orchid culture system for Dendrobium Chao Praya Smile,thin sections (1mm in thickness)of protocorms that developed from seeds served as the starting materials.Thin sections,PLBs and plantlets at the vegetative and transitional stage were all cultured in modi?ed liquid KC medium (Knudson 1946)supplemented with 2%(w/v)sucrose,15%(v/v)coconut water and 4.4m M benzyladenine (BA)on rotary shakers at 120r.p.m.(Yu and Goh 2000a).After the ?oral transition,plant-lets were transferred to the two-layer modi?ed KC medium consisting of a bottom layer of Gelrite-solidi?ed medium and a top layer of liquid medium supplemented with 11.1m M BA as previously reported (Hee et al.2007,Sim et al.2007).All in vitro cultures on liquid or solid medium were kept at 24 C under a 16h photoperiod of 35m mol m –2s –1from daylight ?uorescent lamps.

Wild-type A.thaliana ecotype Columbia-0(Col-0)and 35S:DOSOC1transgenic plants of the same ecotype were grown on soil under long days (16h light/8h dark)at 23±2 C.

Cloning of the DOSOC1cDNA from Dendrobium Chao Praya Smile

Total RNA was isolated from leaves of Dendrobium Chao Praya Smile using the RNeasy ?Plant Mini Kit (Qiagen).Speci?c SOC1-like cDNA fragments were ampli?ed with two degenerate pri-mers,the forward primer (50-GTSCTYTGYGAYGCYGARGT YKCB-30)and the reverse primer (50-NYBNTGYAGYTCYTCA ATNGMRCA-30).A cDNA fragment of around 300bp was ob-tained and cloned into the pGEM-T Easy vector (Promega).After sequencing,the fragment was identi?ed as the partial sequence of a SOC1ortholog.To obtain the full-length se-quence of the cDNA,30-RACE and 50-RACE were performed with gene-speci?c primers 50-CTCCTCGCGGCAAGCTCT ACG-30and 50-CGTCAATCTTCTTTGACATGAGG-30using the SMART TM RACE cDNA Ampli?cation Kit (BD Biosciences Clontech).

Sequence analysis

Alignment of deduced amino acid sequences was made using the Clustal W2multiple sequence alignment program (https://www.wendangku.net/doc/559962913.html,/Tools/msa/clustalw2/)and BOXSHADE 3.21(https://www.wendangku.net/doc/559962913.html,/software/BOX_form.html).The protein sequences of SOC1-like genes aligned in this study were retrieved from the National Center for Biotechnology Information (NCBI)database.The sequences used for phylo-genetic analysis included the MADS domain plus the 110amino acids downstream of the MADS domain as previously reported (Yu and Goh 2000b).The phylogenetic tree was constructed with the Neighbor–Joining algorithm using the software MEGA 4.0

(https://www.wendangku.net/doc/559962913.html,/).

Fig.9Generation of 35S:DOSOC1transgenic orchids.(A)PCR geno-typing using the speci?c primers from 35S and DOSOC1shows the presence of the 35S:DOSOC1transgene in seven independent trans-genic Dendrobium Chao Praya Smile lines that were generated by the MSO selection system followed by the in vitro orchid culture system described in Fig.3.The 35S:DOSOC1plasmid and the genomic DNA from wild-type orchids served as a positive control (C +)and a nega-tive control (WT),respectively.(B)Comparison of ?owering time of wild-type and 35S:DOSOC1transgenic orchids.All seven transgenic lines ?owered (indicated by the ?rst visible in?orescence stalk)earlier than 30wild-type orchid plants examined,among which 16wild-type plants did not produce in?orescence stalks even after 24weeks of culture.

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Expression analysis

Total RNA extracted from either orchid or Arabidopsis served as the template for ?rst-strand cDNA synthesis using the SuperScript ?First Strand Synthesis System (Invitrogen).For semi-quantitative RT–PCR analysis of DOSOC1expression in transgenic Arabidopsis plants,DOSOC1was detected with the primers 50-GCTGGAAGAGCAGGTAGTACAACT-30and 50-GA ATGTGAAAGATCAAGGTCATCC-30,while the b -tubulin gene,TUB2,was ampli?ed as a control with the primers 50-ATCCGTG AAGAGTACCCAGAT-30and 50-TCACCTTCTTCATCCGCA GTT-30.Real-time quantitative PCR was performed in triplicate on the CFX384Real-Time PCR Detection System (Bio-Rad)with the SYBR Green Master Mix (Toyobo).The relative gene expres-sion level was calculated as previously reported (Liu et al.2007).The orchid polyubiqutin gene DOUbi and the Arabidopsis TUB2gene were used as the normalization controls for expression analyses in orchid and Arabidopsis,respectively.Primers used for real-time PCR of genes are as follows:DOUbi ,50-AGGCTAA

GAGGTGGAACAATGATC-30and 50-ATCAGCAAGCTGCTTG CCTGCAT-30;TUB2,50-ATCCGTGAAGAGTACCCAGAT-30and 50-AAGAACCATGCACTCATCAGC-30;DOSOC1,50-CGGC AAGCTCTACGAGTTCT-30and 50-AGCAGGATTCCAGGTTTT CA-30;AGL24,50-GAGGCTTTGGAGACAGAGTCGGTGA-30and 50-CGAGAAGCTGTTCCATTGC-30;and LFY ,50-ATCGCTT GTCGTCATGGCTG-30and 50-GCAACCGCATTGTTCCGC TC-30.

In situ hybridization

Non-radioactive in situ hybridization was carried out as previously described (Yu and Goh 2000b).For synthesis of the DOSOC1antisense RNA probe,the 30end gene-speci?c region of DOSOC1was ampli?ed and cloned into the pGEM-T Easy vector (Promega).The resulting vector was subsequently used as a template for in vitro transcription by the DIG RNA Labeling Kit (Roche Molecular

Biochemicals).

Fig.10Overexpression of DOSOC1in Dendrobium Chao Praya Smile accelerates ?owering.(A)A wild-type orchid plant shows later ?owering than two representative 35S:DOSOC1plants at 16weeks after in vitro culture under our growth conditions.(B)Examination of DOSOC1expression in wild-type and representative 35S:DOSOC1transgenic orchids shown in (A)by semi-quantitative RT–PCR.DOUbi was ampli?ed as a control.Total RNA extracted from leaves was used for this expression analysis.(C)Flower development of 35S:DOSOC1transgenic orchids is either normal (left panel)or abolished (right panel).The arrow indicates a normal ?oral bud.

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Arabidopsis transformation

The full-length cDNA fragment of DOSOC1was cloned into a binary vector pGreen 029-35S (Yu et al.2004)under the control of two CaMV 35S promoters.35S:DOSOC1transgenic plants were generated in the Col background through Agrobacterium tumefaciens -mediated transformation and sub-sequent selection by Basta on soil.

Orchid transformation by particle bombardment

Genetic transformation of Dendrobium Chao Praya Smile was performed utilizing the previously reported MSO selection system (Chai et al.2007)with some modi?cations.

Before bombardment,calli induced from protocorms of Dendrobium Chao Praya Smile were chopped into pieces 3–5mm in diameter,which were pre-cultured for 3d in modi?ed KC liquid medium and then placed on a central core 2cm in diameter on modi?ed KC solid medium on a 90mm diameter Petri dish.The pre-cultured calli were bombarded using the Biolistic PDS-1000/He Particle Delivery System (Bio-Rad).The bombardment chamber was evacuated at a pressure of 700mmHg.Particle bombardment was carried out with a dis-tance of 9cm from the stopping screen to orchid samples at the helium gas pressure of 1,35psi.On average,each bombardment delivered about 8m g of plasmid DNA attached to 1mg of gold particles.

The bombarded calli were maintained on the same plates at 24

C under long-day conditions for 4d for recovery,after which thin sections were transferred to modi?ed KC solid medium con-taining 0.5m M MSO for initial selection of putative transformants.To avoid obtaining chimeric plants,necrotic tissues of calli were removed,while surviving calli were cut into small pieces and subcultured onto fresh solid medium every 2weeks.After three rounds of selection,surviving calli were transferred to modi?ed KC solid medium containing 2m M MSO for lethal selection.Subcultures for removing necrotic tissues and further dividing surviving calli were performed every 2weeks.After another three rounds of selection,proliferated PLBs from surviving calli were identi?ed as putative transformants and continuously cul-tured on modi?ed KC solid medium containing 2m M MSO before they were used for further studies.Putative 35S:DOSOC1transgenic orchids were examined by PCR genotyping using the speci?c primers (50-GACCCTTCCTCTATATAAGGAAGT TC-30and 50-GCTAGAACTGGAGAACTCGTAGAGC-30)that only ampli?ed the fused fragment of 35S and DOSOC1cDNA in the transgene.

Southern blot analysis

A 20m g aliquot of genomic DNA was digested with differ-ent restriction enzymes,resolved on a 0.7%(w/v)agarose gel and then blotted onto a nylon membrane.The blot was hybri-dized overnight with the speci?c digoxigenin-labeled DNA,washed and detected as previously described (Yu and Goh 2000b).

Funding

This work was supported by the Ministry of Education,Singapore [Academic Research Funds (MOE2011-T2-1-018,MOE2011-T2-2-008and R-154-000-506-112)].

Acknowledgments

We thank Dr.Lisha Shen and Dr.Chang Liu for critical reading of the manuscript.

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