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Molecular Regulation of Temperature-Dependent FloralInduction in Tulipa gesneriana1

Hendrika A.C.F. Leeggangers, Harm Nijveen2, Judit Nadal Bigas, Henk W.M. Hilhorst, andRichard G.H. Immink*

Wageningen Seed Laboratory, Laboratory of Plant Physiology, Wageningen University and Research, 6708PBWageningen, The Netherlands

ORCID IDs: 0000-0002-9167-4945 (H.N.); 0000-0002-6743-583X (H.W.M.H.); 0000-0002-0182-4138 (R.G.H.I.).

The vegetative-to-reproductive phase change in tulip (Tulipa gesneriana) is promoted by increasing temperatures during spring. Thewarm winters of recent years interfere with this process and are calling for new adapted cultivars. A better understanding of theunderlying molecular mechanisms would be of help, but unlike the model plant Arabidopsis (Arabidopsis thaliana), very little isknown about floral induction in tulip. To shed light on the gene regulatory network controlling flowering in tulip, RNA sequencingwas performed on meristem-enriched tissue collected under two contrasting temperature conditions, low and high. The start ofreproductive development correlated with rounding of the shoot apical meristem and induction of TGSQA expression, a tulip genewith a high similarity to Arabidopsis APETALA1. Gene Ontology enrichment analysis of differentially expressed genes showed theoverrepresentation of genes potentially involved in floral induction, bulb maturation, and dormancy establishment. Expressionanalysis revealed that TERMINAL FLOWER1 (TgTFL1) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1-like1(TgSOC1-like1) might be repressors, whereas TgSOC1-like2 likely is an activator, of flowering. Subsequently, the floweringtime-associated expression of eight potential flowering time genes was confirmed in three tulip cultivars grown in the field.Additionally, heterologous functional analyses in Arabidopsis resulted in flowering time phenotypes in line with TgTFL1being a floral repressor and TgSOC1-like2 being a floral activator in tulip. Taken together, we have shown that long beforemorphological changes occur in the shoot apical meristem, the expression of floral repressors in tulip is suppressed by increasedambient temperatures, leading either directly or indirectly to the activation of potential flowering activators shortly before thecommencement of the phase change.

The monocotyledonous species tulip (Tulipa gesneriana)originates from Central Asia and grows in mountain-richareas with a temperate climate (Kamenetsky and Okubo,2012; Christenhusz et al., 2013). Most cultivated tulips areproduced in The Netherlands, which has a temperatemaritime climate, fairly resembling the climate of the tu-lip’s region of origin (Compton et al., 2007). The growthcycle of cultivated tulips starts in autumn,when the bulbsare planted in the field. At that moment, all organs, suchas the stem, leaves, and flower, are already present insidethe bulb. A subsequent period of prolonged cold is

required for fast stem elongation as well as for internalpreparation of theflower to bloom in spring (Lambrechtset al., 1994; Rietveld et al., 2000). After this cold winterperiod, the stem elongates, the leaves stretch and unfold,and blooming occurs around April or May, dependingon the cultivar. Themother bulb is completely consumedafter blooming, and themain daughter bulb, also knownas axillarybudA, replaces themother bulb (Botschantzeva,1982). Increasing ambient temperatures in spring areassumed to induce the vegetative-to-reproductive phasechange (floral induction) at the shoot apical meristem(SAM) in the daughter bulb, leading to the developmentof the floral organs and the induction of dormancy(Steward et al., 1971; Gilford and Rees, 1973; DeHertoghand Le Nard, 1993). Once the flower is completely de-veloped inside the bulb, the life cycle starts again.

The morphology of the SAM during floral inductionwas well characterized by Beijer (1952); however, untilnow, the molecular regulation of floral induction hasnot been thoroughly investigated. In contrast, thisprocess has been studied extensively in the model di-cotyledonous species Arabidopsis (Arabidopsis thaliana).In Arabidopsis, floral induction can be triggered bylong days after a period of prolonged cold (vernaliza-tion response), which leads to the down-regulation ofthe flowering repressor FLOWERING LOCUS C (FLC)gene. This repression of FLC facilitates flowering bymaking the SAM sensitive to flower-inducing cues such

1 This work was supported by the Technological Top InstituteGreen Genetics, the Koninklijke Algemeene Vereeniging voor Bloem-bollencultuur, and the Dutch Ministry of Economic Affairs.

2 Present address: Bioinformatics Group, Wageningen Universityand Research, 6708PB Wageningen, The Netherlands.

* Address correspondence to [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Richard G.H. Immink ([email protected]).

H.A.C.F.L. performed the majority of experiments and wrote thearticle with contributions of all authors; H.N. assembled the tran-scriptome and performed the cluster analysis; J.N.B. cloned TgSOC1and performed all yeast two-hybrid experiments; H.W.M.H. pro-vided feedback on the experiments and results; R.G.H.I. supervisedthe research and complemented the writing.

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as ambient temperature and long days (Choi et al., 2011).When the days are getting longer, the photoperiod path-way is induced, leading to the activation of FLOWERINGLOCUS T (FT) by the zinc finger transcriptional regulatorCONSTANS. The perception of changes in the photope-riod is located in the leaves, but floral induction occursat the SAM. In this respect, FT acts as a florigen. The FTprotein is transported via the phloem to the SAM, whereit interacts with the basic leucine zipper (bZIP) transcrip-tion factor FLOWERING LOCUS D (FD). The interactionbetween FT and FD results in the activation of the floralintegrator SUPPRESSOR OF OVEREXPRESSION OFCONSTANS1 (SOC1), SQUAMOSA-BINDING PROTEIN-LIKE (SPL) genes, and finally the flower meristemidentity genes APETALA1 (AP1), LEAFY (LFY), andFRUITFULL (Huijser and Schmid, 2011; Andres andCoupland, 2012).In the absence of the photoperiod pathway in Ara-

bidopsis, the phytohormone GA plays a major role inthe regulation of flowering. GA is known to promotethe expression of SOC1 and LFY, dependent or inde-pendent of the DELLA-mediated pathway, leading tothe activation of the so-called A, B, and C class genes(Lee and Lee, 2010). Together with GA, it is believedthat other endogenous (e.g. other hormones and car-bohydrates) and external (e.g. nutrients and ambienttemperature) signals also play a role in the floral in-duction (Mutasa-Göttgens and Hedden, 2009; Galvãoet al., 2015). The molecular regulation of the phasechange by ambient temperature has been studied to alesser extent in comparison with the vernalization andphotoperiod pathways (for review, see Verhage et al.,2014; McClung et al., 2016). Examples of genes that havebeen associated with ambient temperature-mediatedflowering in Arabidopsis are FLOWERING LOCUS M,SHORT VEGETATIVE PHASE, EARLY FLOWERING3,TERMINAL FLOWER1 (TFL1), and PHYTOCHROME-INTERACTING FACTOR4 (Balasubramanian andWeigel, 2006; Strasser et al., 2009; Kumar et al., 2012; Leeet al., 2013; Pose et al., 2013; Box et al., 2015; Fernándezet al., 2016).Even though it has been shown that flowering time

genes in Arabidopsis are regulated by changes in tem-perature, the change in daylength (photoperiod) is thekey seasonal cue to trigger the reproduction process(Park et al., 1999; Jeong and Clark, 2005; Osnato et al.,2012). In contrast, for tulip, it is assumed that a highambient temperature is the most important seasonalsignal to trigger the floral induction (Khodorova andBoitel-Conti, 2013). In addition, Arabidopsis is a dicotwhile tulip is a monocot, and this large evolutionarydistance may raise the question: how much of the flow-ering time network has been conserved (i.e. how muchknowledge can be transferred fromArabidopsis to tulip)?In the monocotyledonous model species rice (Oryza

sativa), the homologous gene of FT (HEADINGDATE3a)also acts as an activator of flowering, but under short-day conditions (Komiya et al., 2008). This, and manyother examples, reveal that there is at least some simi-larity between dicots and monocots in the molecular

mechanisms underlying flowering time control (Blümelet al., 2015). To date, only a few flowering time genes havebeen identified and characterized in ornamental geo-phytes, such as tulip.A studybyNoy-Porat and colleagues(2013), focusing on daffodil (Narcissus tazetta), identifiedtwo potential flowering time genes with similarity to FTand LFY of Arabidopsis, respectively. In daffodil, NtFTwas shown to be induced by high temperatures at theend of the growth period, correlating with the moment offlowering induction (Noy-Porat et al., 2013). Next to thesesingle-gene approaches, Villacorta-Martin et al. (2015)were, to our knowledge, the first authors to publish agenome-wide study focusing on the vernalization re-sponse and flowering in the ornamental geophyte lily(Lilium longiflorum) by transcriptome profiling.

In this study, a genome-wide approach was under-taken to elucidate themolecular mechanism underlyingthe floral induction and the integration of temperatureresponses in tulip. In The Netherlands, the warm win-ters and high temperatures during spring in recentyears interfered with the floral induction process andinduced an early transition from vegetative to reproduc-tive development, resulting in dehydration of the flower(floral budblasting) or low-quality tulipflowers (vanDamand van Haaster, 2013). This problem calls for the devel-opment of new cultivars that are adapted to this climatechange; hence, detailed molecular knowledge of the pro-cess is required. An experimental setup was designedwith contrary environments, low and high temperature,to identify genes induced by high temperature and theirpossible role in floral induction. RNA sequencing (RNA-seq) was performed to identify differentially expressedgenes in SAM-enriched tissues collected at the differenttemperatures. Subsequently, both a bottom-up and a top-down approach were followed to identify potentialflowering time genes in tulip. For the bottom-up ap-proach, a clustering analysis was performed to obtainan overall picture of the transcriptional changes thatcorrelate with flowering induction, followed by a GeneOntology (GO) enrichment analysis. For the top-downapproach, a direct search based on high similarity withknown flowering time genes was performed. From theidentified potential flowering time genes, eight were fur-ther characterized, and their correlationwith thefloweringtime response was validated in different tulip cultivars.Additionally, heterologous functional analysis of a smallnumber of potential tulip key flowering time regulatorswas performed in Arabidopsis to confirm their proposedrole in the control of this important phase transition.

RESULTS

Morphological Characterization of Floral Induction andEarly Flower Development under High-Temperature Conditions

Tulips bloom in spring, and during developmenttoward blooming the resources in the mother bulb arecompletely consumed. Themother bulb is replaced by asmall number of daughter bulbs including one main

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Tulip Flowering Time Control

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daughter bulb (known as axillary bud A), which iscompetent to flower (Botschantzeva, 1982). The SAMwithin this daughter bulb is present in the middle of thebulb on top of the basal plate and is surrounded byfleshy scales that function as storage organs and pro-vide energy for growth (Fig. 1A; Van der Toorn et al.,2000). The vegetative-to-reproductive phase transitionoccurs in themain daughter bulb shortly after bloomingof the mother bulb and is supposed to be induced byhigh temperatures during spring (Khodorova and Boitel-Conti, 2013). To prove whether temperature is indeed theprimary trigger for the floral induction and to investigatethe process of floral induction at the morphological level,tulip bulbs of cv Dynasty were lifted from the field at theend of spring. The bulbs were transferred to controlled-climate cells with long-day conditions to match with fieldconditions; they were separated into two groups andexposed to low (8°C–9°C) or high (18°C) ambient tem-perature conditions. The temperature courses duringthe growth season in the field and in the climate cellsweremonitored (Supplemental Fig. S1, A and B). Figure1B shows the morphological changes of the SAM thatwere observed in the main daughter bulb at 8°C to9°C in comparison with 18°C. Based on the mor-phological changes of the SAM, Beijer (1952) dividedflower induction and development into seven stages(Supplemental Fig. S2). In order to confirm that floralmeristem identity is indeed established around stageII, the expression of a putative floral meristem iden-tity gene was investigated. In Arabidopsis, AP1 hasbeen identified as a floral meristem identity gene, andthis gene is not expressed during the vegetative stageof development but specifies floral meristems fromthe earliest moment onward (Irish and Sussex, 1990;Sundström et al., 2006). In tulip, two genes belongingto the SQUAMOSA subfamily were identified previouslyin viridiflora tulips and named TGSQA and TGSQB (Hiraiet al., 2010). We isolated these two genes that show highsimilarity with Arabidopsis AP1 and the AP1-like MADSbox gene OsMADS28 of rice (Supplemental Fig. S3;Yamaguchi and Hirano, 2006) from the tulip cv Dynastyand adapted the names TGSQA and TGSQB. The ex-pression of TGSQA was investigated in both low- andhigh-temperature conditions (Fig. 1C). This analysisshows that floral meristem identity is indeed establishedjust after the moment that the SAM enlarges and trans-forms into a dome-like structure,which is approximately6weeks after the start of the high-temperature treatment.This confirms the staging as proposed by Beijer (1952);therefore, the same classification is used in this study.

During the first 5 weeks, under both temperature con-ditions, the SAM of the main daughter bulbs was mor-phologically in stage I and displayed a similar appearance,with one leaf primordium developed and the SAMremaining flat (Fig. 1B). The first morphological dif-ferences between the 8°C to 9°C and 18°C treatmentswere observed from 6 weeks onward. The main daugh-ter bulbs at 8°C to 9°C continued to develop the first leafprimordium and the SAM remained flat, while at 18°C,the SAM started rounding and forming a dome-like

structure (stage II). At 7 weeks, the first floral organprimordium appeared (stage P1) and two additional leafprimordia began to develop.More defined tepal, stamen,and carpel structures were observed after 8weeks at 18°C(stage A2+). In contrast, bulbs at 8°C to 9°C developedone leaf primordium only and the SAM remained vege-tative, even after 8 weeks of low-temperature treatment.Above the soil, the mother plants remained green at thelow-temperature condition (Supplemental Fig. S1C),whereas the mother plants at the high-temperature con-dition senesced completely, resembling the phenotypeunder normal field conditions (Supplemental Fig. S1D).

Transcriptome Analysis during the Floral Induction: ATop-Down Approach

To obtain a better understanding of floral inductionin tulip, transcriptional changes were investigated.RNA-seq was performed on RNA collected from SAM-enriched daughter bulb material collected from week0 (1 d before transfer) up to 7 weeks after the transfer tothe low- or high-temperature environment. Transcriptswere reconstructed de novo using Trinity (Haas et al.,2013). A total of 346,016 transcripts were reconstructed,representing 244,383 Trinity genes (Supplemental TableS1). This large number of putative genes is not unusualwhen using de novo assembly in the absence of a ref-erence genome. In addition, no filtering was used afterthe transcriptome assembly to prevent the loss of se-quence information of lowly expressed transcripts(Moreno-Pachon et al., 2016). The multi-dimensionalscaling (MDS) plot in Figure 1D shows global tran-scriptional changes over time in the SAM at the twodifferent temperature regimes. In the low-temperaturecondition, very few morphological changes are occur-ring in the SAM inside the bulb (Fig. 1B), which is ac-companied by only a few transcriptional changes. Incontrast, gene activity in bulbs in the high-temperaturecondition is changing substantially over time. The samplestaken from week 2.5 until week 6 cluster together, whilesamples from 7 weeks after the transfer form a separatecluster. This clustering reveals that high temperatureshave an immediate effect and that floral induction, andlikely other high-temperature-induced processes, are af-fected directly from the start of the temperature treatment(week 2.5). Subsequently, based on global transcriptionalchanges, the bulbs remain in this stage for several weeks,followed by a second change in global expression atweek 7. This later burst of differential expression coincidesperfectly with the morphological changes of the SAM(Fig. 1B) and the induction of flowering, as confirmedby the increase of TGSQA transcript abundance (Fig. 1C).

For further identification of putative flowering time-controlling genes and to gain insight into the globaltranscriptional changes, an initial top-down approachwas followed (Leeggangers et al., 2013). The top-downapproach consisted of an untargeted analysis using GOenrichment and a clustering analysis. In the GO en-richment analysis, genes differentially expressed upon

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high-temperature treatment were selected to get anindication of the biological processes affected by thistreatment. For this purpose, transcript abundance ateach interval was compared with the situation at themoment just before the transfer to controlled environ-mental conditions (week 0). Figure 2 displays a selec-tion of GO terms that were found to be overrepresentedin the significantly up- and down-regulated genes athigh ambient temperature but not in low-temperatureconditions at the same time point. A more complete

overview of overrepresented GO terms can be foundin Supplemental Figure S4. As expected, the panel ofup-regulated genes contains GO terms related to theflowering process, such as the regulation of flower de-velopment and vegetative-to-reproductive phase tran-sition of the meristem. These GO terms corroborate themorphological changes (Fig. 1B). Besides these directlyflowering-relatedGO terms, several others, such as sugar-mediated signaling pathway, cell cycle, response to tem-perature stimulus, and RNA splicing, are connected with

Figure 1. Morphology of the vegetative-to-reproductive phase change at the SAM and transcriptional changes over time. A,Morphology of the SAM inside the bulb and its surrounding tissues in spring prior to the temperature experiment. Note that theSAM is still vegetative and that one leaf primordium has developed. Bar = 1 mm. B, Morphological changes at the SAM inside themain daughter bulbs of cv Dynasty during low- and high-temperature conditions. In the first 5 weeks of both temperatureconditions, only one leaf primordiumdeveloped (green) and the SAM remained flat (yellow). After 6 weeks at 18°C, the SAMgot adome-like appearance, which is the first known morphological change upon making the switch from vegetative to reproductivedevelopment. Shortly after this, the floral meristem (FM; orange) gives rise to the development of the different floral organs(tepals, cyan; stamens, violet; carpel, red). Note that the SAM of bulbs in the low-temperature condition (8°C–9°C) remainsvegetative and flat for the complete period of 8weeks. The different tissues have been artificially colored in the right image of eachgroup. Bars = 1 mm. C, Expression pattern of TGSQA at low-temperature (8°C–9°C) and high-temperature (18°C) conditions. D,MDS plot revealing global transcriptional changes over time. The bulbs from the low-temperature (8°C–9°C) condition remain ina relatively stable transcriptional state, whereas the bulbs from the high-temperature (18°C) condition show significant tran-scriptional changes over time associated with the switch from the vegetative to the reproductive phase. C, Cold; W, warm; thenumber indicates the week after the start of the experiment. FC, Fold change.






the vegetative-to-reproductive phase transition. Oneexample of sugar-mediated signaling involved in theflowering process is trehalose-6-phosphate signalingin Arabidopsis, for which the gene TREHALOSE-6-PHOSPHATE SYNTHASE1 is required for the timingof the initiation of flowering (Wahl et al., 2013). Also,the process of RNA splicing has been shown to play arole in ambient temperature-mediated flowering timecontrol in Arabidopsis (Verhage et al., 2014; Capovillaet al., 2015).

In addition to direct flowering-related GO terms, otherGO terms related to plant metabolism are overrepre-sented in both groups of up- and down-regulated genes.One example is carbohydrate biosynthetic process,which is found to be overrepresented in the up-regulatedgenes, while carbohydrate metabolic process is overrep-resented in the down-regulated genes. The up-regulatedgenes are mostly present in secondary metabolite bio-synthesis and glycan biosynthesis/metabolism, whilethe down-regulated genes are mostly present in lipidmetabolism (fatty acid biosynthesis) and metabolism of

other amino acids. In this respect, it is good to realize that,at the same moment that a decision is made to flower ornot to flower, parts of the mother bulb (e.g. leaves, stem,and scales) are senescing and the daughter bulbs matureand become dormant (De Hertogh and Le Nard, 1993).Therefore, it is possible that the overrepresentation ofthese metabolism-specific terms is not, or not only, re-lated to floral induction but also to these physiologicalchanges.

The GO terms overrepresented in the down-regulatedgenes in the high-temperature condition are mostly re-lated to metabolic processes such as amine metabolismand alcohol metabolism, but also hormone related, suchas brassinosteroid biosynthesis and jasmonic acid me-tabolism. Thus, the GO enrichment analysis revealedthat, among the up-regulated differentially expressedgenes, flowering-related GO terms are present togetherwith GO terms related to bulb maturation and the in-duction of dormancy.

As a second top-down approach, a coexpression clus-tering analysis of all transcription factors was performed

Figure 2. Overview of the GO enrichment analysis in the transcriptome data of the floral induction in tulip. Output GO en-richment analysis was performed by comparing each week with week 0. At left in red, the GO terms listed are specificallyoverrepresented in the up-regulated genes upon high temperatures. At right in blue, GO terms specifically overrepresented in thedown-regulated genes are shown.


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to focus specifically on regulatory genes for which theexpression correlates with high-temperature-inducedfloral induction in tulip and corresponding morpho-logical changes. Of the clusters with an expressionpattern that can be related to floral induction, threeselected clusters contain at least one gene showinghigh sequence similarity with an Arabidopsis flower-ing time regulator (Fig. 3; Supplemental Fig. S5). Incluster 17, a transcript showing high similarity withthe floral repressor AP2 (Jofuku et al., 1994) is presentthat shows a steep drop in expression after week 4 (Fig.3A). This is approximately 2 weeks before the SAMobtains its characteristic dome-like structure. In addi-tion to AP2, also a transcript showing high similaritywith the floral repressorALB3 (Wang andWang, 2009)is present in this cluster. Other putative transcriptionfactor genes in this cluster are ATIPS2, ARF22, andJAZ1. Although these genes are not associated directlywith flowering in Arabidopsis, their expression patternssuggest a relation with the repression of flowering. Fur-ther detailed analyses are needed to explore possible rolesof these regulatory genes in the flowering time response.Cluster 37 contains a transcript showing high similaritywith the Arabidopsis ATC gene, another repressor offlowering (Huang et al., 2012). Its expression decreasedgradually until week 5 (Fig. 3B). The fact that bothclusters 17 and 37 contain putative flowering repressorssuggests that the block on flowering is removed aroundweek 4 after high-temperature induction. Other tran-scripts present in cluster 37 have been related in Arabi-dopsis to trichromebranching and seed coat development

(MYB5; Li et al., 2009), cell wall biosynthesis (GAUT15;Persson et al., 2007), and lignin biosynthesis (PRR2;Nakatsubo et al., 2008). Finally, in cluster 238, two tran-scripts showing high similarity with known floweringtime functions inArabidopsis were present (Fig. 3C). Thefirst is the flowering time gene FLK, which acts as a re-pressor of FLC in Arabidopsis (Mockler et al., 2004). Thesecond gene is FT, which acts in Arabidopsis as a floralintegrator (Yamaguchi et al., 2005). The expression ofthese putative flowering time genes increased steadilyfrom week 0 onward until they reached a plateau ofmaximum expression around week 4. This interestingcluster also contains the genes TBP2 and EDA35. It isattractive to attach a potential function as a floweringinducer to these genes, but it is good to realize that, atthe same moment during development, other biolog-ical processes are active to which these genes might berelated. Hence, we cannot exclude that their correla-tion with morphological flowering is coincidental.

Identification and Characterization of Putative FloweringTime Genes: A Bottom-Up Approach

The top-down approach provided first insights intothe flowering time gene regulatory network and pointedtoward genes potentially involved in a variety of high-temperature-induced biological processes, includingfloral induction. However, it also revealed limitationsof the identification of key regulatory genes of a singledefined process based solely on expression association.Therefore, a bottom-up approach was followed as well,

Figure 3. Three selected clusters from the cluster analysis of all tulip transcripts that have high similarity with known transcriptionfactors in Arabidopsis. The clusters represent transcripts of the high-temperature condition.On the x axis, the different time pointsare plotted, and on the y axis, the z score is shown (normalized cpm). A, Expression of the genes in cluster 17 remains stable untilweek 4, after which their expression decreases. The cluster includes AP2, INOSITOL 3-PHOSPHATE SYNTHASE2 (ATIPS2),AUXIN RESPONSE FACTOR22 (ARF22), JASMONATE-ZIM-DOMAIN PROTEIN1 (JAZ1), andALBINO3 (ALB3). B, Expression ofthe genes in cluster 37 decreases slowly over time. This cluster includes ARABIDOPSIS CENTRORADIALIS (ATC ), MYB5,GALACTURONOSYLTRANSFERASE15 (GAUT15), and PINORESINOL REDUCTASE2 (PRR2). C, Expression of the genes incluster 238 increasing from week 0 onward and reaching a plateau around week 4. This cluster includes FLOWERINGLOCUS K (FLK ), FT, TATA BOX-BINDING PROTEIN2 (TBP2), and EMBRYO SAC DEVELOPMENT ARREST35 (EDA35).Transcripts with a high similarity (BLAST cutoff value of 1e-05) to a known flowering time gene in Arabidopsis are markedwith red stars.






guided by the wealth of knowledge on the molecularnetwork of flowering time control in Arabidopsis. In thismodel species, over 170 genes have been identified anddescribed that are known to play a role in flowering timecontrol (Fornara et al., 2010).

For 57 Arabidopsis flowering time genes, one ormore sequences with high similarity could be identifiedin tulip (Supplemental Fig. S6). Based on the observedexpression patterns in tulip during high-temperature-induced flowering, eight geneswere selected for furtherdetailed studies of their proposed functioning in flow-ering induction. To confirm their expression pattern,as well as the overall quality of our RNA-seq assemblyand differential gene expression analysis, the expres-sion patterns of these selected genes were confirmed byquantitative reverse transcription PCR (Fig. 4). Amongthese eight genes is a gene with high similarity to thefloral repressor TFL1; therefore, it was designated TgTFL1(Supplemental Fig. S7A). In the high-temperature condi-tion, the expression of this gene decreased instantlyafter the start of the treatment, while under the low-temperature treatment, transcript abundance de-creased gradually but slowly over the whole period of8 weeks (Fig. 4A). A similar pattern was observed forthe gene belonging to the TM3 subfamily TgSOC1-like1(TgSOC1L1; Fig. 4B, Supplemental Fig. S7B). Based onthese expression patterns, both genes seem to act asrepressors of flowering. For TgTFL1, this is expected,based on Arabidopsis data (Hanano and Goto, 2011),but for TgSOC1L1, this is a surprising observation,taking into account the function of the floral integratorAtSOC1 (Lee and Lee, 2010). However, besides this

TgSOC1L1 gene, another member of the TM3 sub-family clade was identified and named TgSOC1-like2(TgSOC1L2; Supplemental Fig. S7B). Expression ofTgSOC1L2 increased between weeks 4 and 6 and de-creased again between weeks 6 and 7 in the high-temperature condition (Fig. 4C). AtSOC1 showed asimilar increase in expression toward the vegetative-to-reproductive phase change, after which its expressiondiminished during further flower and floral organ de-velopment (Lee and Lee, 2010). Another potential floralintegrator that could be identified is homologous toArabidopsis FT, designated TgFT-like (SupplementalFig. S7B). The abundance of this TgFT-like transcriptalso increased from week 4 onward, but instead of adecrease in expression, like TgSOC1L2, its expressionwas induced throughout the whole measured period(Fig. 4D). In the low-temperature condition, both geneswere not expressed; hence, for both genes, a positivecorrelation with floral induction was observed, pro-viding evidence that these genes might act as activa-tors of flowering in tulip.

Morphological data and the expression of the AP1-like gene TGSQA in tulip (Fig. 1C) suggested thatflower development starts during week 6, and theexpression of the putative floral organ identity geneSEPALLATA1 (SEP1) and TGSQB correlates with this(Fig. 4, E and F; Supplemental Fig. S6). Furthermore,these geneswere not expressed in the low-temperaturecondition in which the SAM remains vegetative.TGSQA has a high similarity to AP1 from Arabidopsisand the snapdragon (Antirrhinum majus) SQUAMOSAgene (Supplemental Fig. S3). SQUAMOSA genes are

Figure 4. Expression analysis by quantitative reverse transcription-PCR of eight putative tulip flowering time genes in the SAMregion of the main daughter bulb during 8 weeks of high- or low-temperature treatment. A, Expression of TgTFL1. B, Expressionof TgSOC1L1. C, Expression of TgSOC1L2. D, Expression of TgFT-like. E, Expression of TgSEP1. F, Expression of TGSQB. G,Expression of TgSPL1. H, Expression of TgSPL2.


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regulated by SQUAMOSA PROMOTOR-BINDINGPROTEIN box genes (Preston and Hileman, 2010), ofwhich two were identified in the tulip transcriptome,designated TgSPL1 and TgSPL2. Both genes have dif-ferent expression patterns (Fig. 4, G and H). TgSPL2might act as a floral repressor, because its expressiondecreased from weeks 0 to 4 under high-temperatureconditions but increased under the low-temperaturecondition. In contrast, TgSPL1 was induced specifi-cally by high temperature, and this increase coincidedwith the up-regulation of TGSQA, making it a putativecandidate for this regulatory function upstream ofTGSQA and suggesting a conservation of this regula-tory link between Arabidopsis and tulip.

Genetic Diversity as a Tool to Confirm the Role of PutativeTulip Flowering Time Genes

To date, no efficient tools are available to transformTulipa spp. (Kanno et al., 2007). Therefore, genetic di-versity was used as a tool to obtain additional confir-mation of the proposed role of a selection of genes in theflowering time response. Second, the potential tulipflowering time regulators were identified under con-trolled temperature conditions in climate cells; there-fore, the experiment was repeated with the originalcultivar (cv Dynasty) and selected additional cultivarsunder their natural conditions in the field. Unfortu-nately, no detailed information is available about themoment of floral induction in different tulip geneticbackgrounds. However, the moment of blooming inspring has been reported for a large number of tulipcultivars, and we hypothesized that there is a direct cor-relation between the timing of blooming and themomentof the vegetative-to-reproductive phase change insidethe main daughter bulb. Initially, six tulip cultivars wereselected with variable blooming times in spring(Supplemental Fig. S8A). After blooming of the motherbulb, bulbs of all cultivars were lifted at the same time.From1month before lifting until 8weeks after lifting, themorphological changes related to the floral inductionwere monitored (Supplemental Fig. S8B). Surprisingly,earliness in the floral induction appeared not to be cor-related with early blooming in spring. One of the latest-blooming cultivars (cv Strong Gold) of our selection wasshown to be one of the first making the developmentalswitch from the vegetative to the reproductive phase.However, it is important to note that cv Strong Gold isknown to be a temperature-sensitive cultivar. The dif-ferences in the timing of the phase change appeared to belimited to approximately 1 to 2 weeks only, and all cul-tivars reached stage P1 (first whorl of tepals formed)almost at the same time (Supplemental Fig. S8B). Afterreaching this stage, the floral buds developed at a similarspeed. Based on these observations, the most diversifiedcultivars in the moment of the floral induction were se-lected for molecular analysis (cv Strong Gold, early; cvPurple Prince,mid; cvDynasty, late; Fig. 5A). From thesethree cultivars, cv Purple Prince and cv StrongGold haveone parent in common (cv Yokohama).

Expression of the eight selected putative floweringtime genes was monitored in the three cultivars (Fig. 5,B–I). The expression ofTgTFL1decreasedfirst in cv StrongGold, starting from 4 weeks before lifting, followed 1 to2weeks later in cv Purple Prince and cvDynasty (Fig. 5B).In the case of TgSOC1L1, the expression in all three culti-vars decreased in a similar manner (Fig. 5C). The samewas observed for the putative floral inducer TgSOC1L2,the expression of which increased from 1 week beforelifting (week21) in all three analyzed cultivars and, afterreaching a high steady-state level, started to decreaseslowly after the transition to reproductive development(Fig. 5D). Also, TgFT-like expression increased 1 weekbefore lifting (week 21) in all cultivars (Fig. 5E). The ex-pression of TgSEP1 and TgSPL2 was similar for all culti-vars and increased from 3weeks after lifting onward (Fig.5, G and I). For the TGSQA and TgSPL1 genes, a slightlyearlier induction was observed in cv Strong Gold incomparison with cv Dynasty, which is in line with theearlier floral induction in this cultivar (Fig. 5, F and H).

In conclusion, all selected genes showed similar behaviorin expression pattern in this field experiment performedin 2015 to the previous controlled-climate chamber experi-ment in 2013. The observed expression patterns and levelswere in line with the supposed functions of the analyzedgenes in flowering time control and, as such, providedadditional evidence for their proposed roles in this bio-logical process. Whereas for some of the genes no differ-ences in expression could be observed at the exactmomentof repression or induction in the three cultivars, TgTFL1,TGSQUA, andTgSPL1 showed expression changes tightlylinked to the small differences in the timing of the phaseswitch from vegetative to reproductive development.

Heterologous Expression of Tulip Flowering Time Genesin Arabidopsis

To further investigate the functions of two selectedpotential tulip flowering time regulators, heterologousoverexpression studies were performed in Arabidopsis.Transgenic Arabidopsis lines in which the tulip genesTgSOC1L2 and TgTFL1were placed under the control ofthe constitutive cauliflower mosaic virus 35S promoter(Odell et al., 1985) were generated and phenotyped forflowering time. Overexpression of TgSOC1L2 resulted ina weak early-flowering phenotype (Fig. 6, A and E–G),while overexpression of TgTFL1 resulted in a severe late-flowering phenotype (Fig. 6, B and H–J). In addition tothe late-flowering phenotype upon overexpressingTgTFL1, floral organ morphological changes were ob-served that are similar to those observed when ectopi-cally expressing AtTFL1 (Fig. 6, C and D; Shannon andMeeks-Wagner, 1991), confirming that TgTFL1 is similarin function and behavior to AtTFL1.

Protein Interaction Partners of Potential Tulip FloweringTime Regulators

For both AtSOC1L2 and AtTFL1, protein-protein inter-action studies have been reported (de Folter et al., 2005;








van Dijk et al., 2010; Hanano and Goto, 2011) that pro-vide information about their biological and molecularfunctions. In total, 25 protein-protein interactions be-tween AtSOC1 and other MADS domain transcriptionfactor proteins were reported in the study of de Folter

and colleagues (2005). To test whether the tulip ho-molog TgSOC1L2 is able to interact with the same setofMADSdomain proteins asAtSOC1, the protein-proteininteraction between TgSOC1L2 and the collection ofArabidopsisMADS domain proteinswas studied. Yeast

Figure 5. Morphological and molecular analysis of the vegetative-to-reproductive phase change in three tulip cultivars. A, Morpho-logical analysis of the changes at the SAM in cv Purple Prince, cv Dynasty, and cv Strong Gold. I, Vegetative; II, reproductive; P1, firstwhorl of tepals; P2, secondwhorl of tepals; A2, secondwhorl of stamens; A2+, beginning of carpel development. FM, Floral meristem.The first visual observation of the transition from vegetative to reproductive development is marked with an asterisk. Bars = 1 mm. B,Expression of TgTFL1. C, Expression of TgSOC1L1. D, Expression of TgSOC1L2. E, Expression of TgFT-like. F, Expression of TGSQA. G,Expression of TgSEP1. H, Expression of TgSPL1. I, Expression of TgSPL2.

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Copyright © 2017 American Society of Plant Biologists. All rights reserved.


two-hybrid analyses revealed that TgSOC1L2 is able tointeractwith 18ArabidopsisMADSdomain proteins (Fig.7A), of which, remarkably, only four are in commonwithAtSOC1 (AGAMOUS-LIKE12 [AGL12]/XAANTAL1,AGL17, AGL19, and AGL44/ARABIDOPSIS NITRATEREGULATED1). This difference in protein-protein inter-action pattern between AtSOC1 and TgSOC1L2 mightexplain the weak early-flowering phenotype upon over-expressing TgSOC1L2 in Arabidopsis (Fig. 6). The lack ofinteraction with the classical ABC-class proteins alsocould explain why no flower phenotypes appearedupon ectopic expression of TgSOC1L2, in contrast towhathas been found when ectopically expressing AtSOC1 inflowers (Borner et al., 2000). In AtSOC1, certain interac-tion motifs have been characterized and found to be

required for protein-protein interactions (van Dijk et al.,2010). When aligning the TgSOC1L2 and AtSOC1 pro-tein sequences, mutations are present at almost all thesemotifs supposed to be important for protein-protein in-teractions, except formotif 2 (Supplemental Fig. S9). Thissupports the difference observed in the protein-proteininteractions of TgSOC1L2. Overexpression of TgTFL1 inArabidopsis gave a similar phenotype to overexpressionof AtTFL1 (Ratcliffe et al., 1998). It is known that, inArabidopsis, both AtFT and AtTFL1 can interact withthe bZIP transcription factor FD (Hanano and Goto,2011). To test whether TgTFL1 is able to interact withAtFD, a yeast two-hybrid assay was performed. TgTFL1showed interaction with AtFD and AtFDP (Fig. 7B),suggesting that, similar to AtTFL1, TgTFL1 can interfere

Figure 6. Phenotypic and molecular analyses of Arabidopsis overexpressing different potential tulip flowering time genes. A, 35S:TgSOC1L2. B, 35S:TgTFL1. C,Wild-typeflower.D,35S:TgTFL1flower. E,Number of days to flowering for 35S:TgSOC1L2. F, Leaf numberof 35S:TgSOC1 when the inflorescence reaches a length of 1 cm. G, Expression of TgSOC1L2 in the overexpression line TgSOC1L2-2in comparison with Columbia-0 (Col-0). H, Number of days to flowering for 35S:TgTFL1. I, Leaf number of 35S:TgTFL1 when the in-florescence reaches a length of 1 cm. J, Expression of TgTFL1 in the overexpression line TgTFL1-1 in comparison with Columbia-0.* represents,0.05 significance and ** represents,0.01 significance.






with the FT/FD-dependent transcriptional activation offlowering time (Hanano and Goto, 2011).

DISCUSSION

In this study, a deep-sequencing RNA-seq approachwas followed to shed light on the transcriptional changesoccurring prior to and during the switch from vegetativeto reproductive development in the bulbous plant speciestulip. A broad range of in silico analyses and confirmationof observed expression patterns by quantitative reversetranscription-PCR provided strong evidence for a set oftulip genes to represent regulators offlowering. For two ofthe identified genes, their supposed roles as repressor andactivator of flowering, respectively, could be confirmedby heterologous functional analyses in Arabidopsis. Weshowed that high ambient temperatures are inducingflowering in tulip and that transcriptional changes asso-ciatedwith the flowering time response occur already 4 to5 weeks before the first flowering-related morphologicalchanges of the SAM become visible.

Flowering Induction Cooccurs with Bulb Maturation andthe Initiation of Dormancy

Simultaneouslywith flower initiation in the SAM, thedaughter bulbs mature and are prepared for a period ofdormancy (De Hertogh and Le Nard, 1993). In line withthese developmental and physiological conditions, weidentified an overrepresentation of GO terms such asdormancy process, seed maturation, and response toabscisic acid (ABA) in the GO analysis of the differen-tially expressed genes. The phytohormone ABA is oftenassociated with the establishment and maintenance ofseed dormancy (McCarty, 1995). Seeds are preventedfrom precocious germination by the presence of ABA,the osmotic environment, and, possibly, by limiting theavailability of energy and nutrients (Garciarrubio et al.,

1997; Bewley et al., 2013). In the transcriptome data oftulip, something similar is observed, as many genes an-notated with metabolite-associated GO terms, such asamine metabolic process and carbohydrate metabolism,are down-regulated in the meristem-enriched tissue col-lected from tulip. Down-regulation of metabolism likelyis associated here with the preparation or establishmentof dormancy, very similar to what is observed in seeds.Thus, based on the transcriptome, maturation and prep-aration for dormancy in tulips resembles the process ofmaturation and dormancy induction in seeds. In addi-tion, several studies have shown that ABA can eitherinhibit or promote flowering, depending on the species(Wang et al., 2002, 2013; Frankowski et al., 2014). InArabidopsis, the bZIP transcription factor ABSCISICACID-INSENSITIVE MUTANT5 is involved in the re-pression of the floral transition by up-regulation of thevernalization-responsive gene FLC (Wang et al., 2013).Also, in Pharbitis nil (Japanese morning glory), ABA hasbeen shown to have an inhibitory effect on flowering,likely through the modulation of ethylene biosynthesis(Frankowski et al., 2014). In contrast to Arabidopsis andP. nil, in which ABA inhibits flowering, ABA promotesflowering in Litchi chinensis (lychee nut). In this species,the application of exogenous ABA promoted flowering,and this was impaired by the expression of LcAP1, thehomolog ofArabidopsisAP1 (Cui et al., 2013). Thus,ABAhas been associated with several biological processes,ranging from metabolic arrest, dormancy initiation, andtissue maturation to the control of flowering time. Obvi-ously, more research is required to pinpoint the exactfunction of ABA during floral bud initiation in tulip.

Functioning of a Tulip TFL1 Gene as a PotentialFlowering Repressor

We have identified TgTFL1 as a potential inhibitor offlowering in tulip. Down-regulation of its expression

Figure 7. Yeast two-hybrid assayof potential tulip flowering timeregulators. A, Protein-protein in-teractions between TgSOC1L2and Arabidopsis MADS domainproteins (synthetic drop-out me-dium– Leu, Trp, andHis + 1mM3-aminotriazole). B, Protein-proteininteraction of TgTFL1 and Arabi-dopsis FD and FDP proteins (syn-thetic drop-outmedium– Leu, Trp,and His + 1 mM 3-aminotriazole).AtFT, AtTSF, and AtTFL1 wereadded to the assay as positivecontrols. AD, Activation domain;BD, binding domain.


Leeggangers et al.



appears to be temperature dependent and is initiated4 to 5 weeks prior to the switch to reproductive de-velopment. In the dicot Arabidopsis, TFL1 also acts asa flowering repressor and has been proposed tofunction in the ambient temperature pathway as a hubbetween the photoperiodic and ambient temperatureflowering time signaling (Strasser et al., 2009). Also, instrawberry (Fragaria vesca), the AtTFL1 homolog FvTFL1integrates photoperiod and temperature signals in orderto repress flowering (Rantanen et al., 2015). Differentfrom Arabidopsis and strawberry, tulip is a day-neutralplant; therefore, photoperiod is not supposed to play arole in the regulation offlowering time (Kamenetsky andOkubo, 2012). However, surprisingly, GO terms relatedto photoperiod, such as photoperiodism and response tolight stimulus, were found to be overrepresented in thegenes up-regulated by high flowering-inducing ambienttemperatures, and overall, their expression patternscorrelated perfectly with the genes belonging to theGO category vegetative-to-reproductive phase tran-sition of meristem. A bulb is an underground plantstructure, but when these specific genes are induced,the plants still have green leaves aboveground thatmay translate a light or photoperiodic signal to theSAM in the daughter bulbs. It will be of great interestto investigate whether the observed expression dif-ferences of photoperiodic genes plays a role in theinduction of flowering in tulip and whether thefunction of TFL1 as an integrator of the photoperiodand ambient temperature pathways is conserved inmonocots and in bulbous plant species such as tulip.The life cycles of tulip and the short-day plantstrawberry have a lot in common. Under short-day

and low-temperature conditions in autumn, strawberrymakes the transition of the vegetative to reproductivephase change. Then, after the winter period, the flowersemerge and blooming occurs in spring (Koskela et al.,2012). FvTFL1 is a strong regulator controlling theseasonal flowering of strawberry (Rantanen et al., 2014).Not only in strawberry, but likely also in other peren-nials, including tulip, TFL1-like genes play an impor-tant role in the timing of flowering and the duration ofblooming.

TgTFL1 is of interest not only in relation to floweringtime control but also to the function of AtTFL1 inmaintaining inflorescence meristem identity and, assuch, is essential for indeterminate inflorescence de-velopment and the production of multiple flowers. Amutation in the Arabidopsis TFL1 gene transforms theindeterminate inflorescence into a terminal floral meri-stem (Shannon and Meeks-Wagner, 1991), which issimilar to tulip reproductive stage morphology. Thedifference between these two species most likely canbe explained by the fact that, in contrast with Arabi-dopsis, TgTFL1 expression remains low throughoutflower development following its reduction towardthe phase switch. Nonetheless, when overexpressingTgTFL1 in Arabidopsis, not only is flowering delayedbut also petals of most flowers are absent, suggestingthat TgTFL1 in Arabidopsis is able to repress AtAP1.This shows that the sequences of TgTFL1 and AtTFL1are sufficiently similar and conserved to maintain thisfunction in the repression of AtAP1 and that terminalflower formation in tulip is most likely not due to amutation in the TgTFL1 protein but to a mutation ofthe TgTFL1 expression pattern.

Figure 8. Proposedmodel of the vegetative-to-reproductive phase change in tulip. During spring, high temperature induces the floral induction in tulipby first repressing TgTFL1 and TgSOC1L1. After this suppression, the floral activators TgSOC1L2 and TgFT-like are induced, leading to direct or indirectactivation of floral meristem and organ identity genes (TGSQA, TGSQB, and TgSEP1).





Existence of TgSOC1-Like Genes with PossibleAntagonistic and Novel Functions in Flowering

Two SOC1-like genes were identified in tulip that,surprisingly, appeared to respond in an opposite mannerto the temperature treatments. The gene that we desig-nated TgSOC1L1 has an expression pattern typical for aflowering repressor, whereas TgSOC1L2 resembles theexpression of AtSOC1 and that of a flowering inducer. Inline with this observation, overexpression of TgSOC1L2in Arabidopsis caused a weak but significant early-flowering phenotype. In this respect, it should be re-alized that the tulip’s life cycle is different from the lifecycle of Arabidopsis (Anderson, 2006; Sofo, 2016). Thisdifference is not only in duration but also when thetransition to the reproductive phase is made and bloom-ing occurs. In Arabidopsis, flowering commences directlyupon the switch from vegetative to reproductive devel-opment,whereas tulipflower buds become dormant aftertheir initiation and still require a period of prolonged coldin order to bloom in spring (Lambrechts et al., 1994). Wefound that TgSOC1L2has a dimerizationpattern differentfrom AtSOC1 in the heterologous yeast two-hybridscreen. One of the striking differences is that TgSOC1L2interacts withmembers of the FLC/MADSAFFECTINGFLOWERING (MAF) clade, which are known for theirfunction in vernalization and ambient temperature flow-ering time responses. Therefore, it seems that TgSOC1L2evolved different functions in comparison with AtSOC1but seems to be a key regulator of the ambient temperature-controlled flowering time in tulip.

CONCLUSION

This study has confirmed that high temperature isan important trigger of the vegetative-to-reproductivephase transition in tulip. A large number of potentialflowering time regulators have been identified thatpartially appear to be conserved when compared withthe dicot Arabidopsis. Our results are summarized in aputativemodel of themolecular regulation of the floralinduction in tulip (Fig. 8). We have identified a largenumber of potential novel flowering time regulators thatmight be bulbous plants or tulip specific. The initiation offlower development, maturation of the bulb, and estab-lishment of dormancy all take place at the samemoment.Therefore, it is of great interest to study the interactionsbetween these processes in more detail and to resolve thecomplexity of events occurring in daughter bulbs, whenfrom the outside nothing seems to be happening and thebulbs are establishing summer dormancy.

MATERIALS AND METHODS

Plant Material and Growth Conditions

Tulip (Tulipa gesneriana ‘Dynasty’; size 10/11) bulbswere planted in crates inthe field in early November 2012 and transferred to two different controlledtemperature conditions, 8°C to 9°C and 19°C, at the beginning of June 2013 afterdecapitation of the flower. The bulbs were planted in crates to prevent damage

to the roots by transfer to the temperature-controlled climate cells. The bulbswere maintained in the climate cells at the two indicated temperature regimeswith a 16-h photoperiod, with 100 mmol s21 m22 light, for 9 weeks. A mix ofmeristem-enriched tissues (square cutting of 0.5 3 0.5 cm including the meri-stem and leaf primordia; Fig. 1A), derived from five individual tulip bulbs, wasdissected with a scalpel and pooled together to form one biological replicate,and this was repeated three times, once every week in the afternoon (CentralEuropean Time, 1–3 PM). Each mix of meristem-enriched tissue was homoge-nized by the use of liquid nitrogen, mortar, and pestle and stored at280°C untiluse. In addition to artificially stimulating or preventing the floral induction bycontrolled temperature conditions, six cultivars (cv Northgo, cv Purple Prince,cv Dynasty, cv Ile de France, cv Strong Gold, and cv Yellow Flight) wereplanted directly in the field at the end of October 2014 and harvested in June2015. After harvest, the bulbs were placed at 25°C for 10 d to dry. After these10 d, the bulbs were stored at 17°C to 20°C in the dark. Samples of meristem-enriched tissue were collected during the cycle as described above and storedat 280°C until use.

Arabidopsis (Arabidopsis thaliana) seedswere stratified for 2 to 3 d at 4°C andgerminated, and a segregating plant population (30–50 plants) was grownunder long-day conditions (16/8 h of light/dark) at 20°C on Rockwool blocks.Flowering time was scored by counting the number of rosette leaves at themoment the inflorescence reached a length of 1 cm.

Microscopic Imaging

Morphological changes of the SAM region inside the bulb during the veg-etative and reproductive phases were monitored with a Carl Zeiss SV11 ste-reomicroscope (Zeiss), and photographs were taken with a Nikon digital sightDS-Fi1 camera (Nikon).

Total RNA Extraction and cDNA Synthesis

To extract the total RNA of meristem-enriched tissue, the Tripure protocol(Roche)wasused according to themanufacturer’s instructionswith the additionof 2% (w/v) polyvinylpyrrolidone and 2% (v/v) b-mercaptoethanol to theextraction buffer. Subsequently, a DNase treatment with RQ1 (Promega) wasperformed to remove DNA, followed by a phenol:chloroform (1:1) extractionand ethanol precipitation. A total amount of 500 ng was used for first-strandcDNA synthesis using Moloney murine leukemia virus reverse transcriptase(Thermo Scientific) following the protocol from the manufacturer and oligo(dT)primers. All reactions were performed in a MyCycler (Bio-Rad).

Strand-Specific RNA-seq

Total RNAofmeristem-enriched tissues, collected between June and lateJuly 2013, was used for RNA-seq. For the preparation of the RNA-seqcDNA library, the TruSeq stranded mRNA sample preparation kit (Illumina)was used according to the manufacturer’s instructions. The quality of the li-braries was examined with the Bioanalyzer 2100 DNA 1000 chip (AgilentTechnologies). The Illumina HiSeq2000 platform was used to obtain 100-bppaired-end reads.

RNA-seq Data Analysis

The quality of the reads obtained from RNA-seq was examined by FastQC(http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Trimming ofthe reads by Trimmomatic version 0.32 (Bolger et al., 2014) was used to improvetheir quality. After trimming, a de novo assembly was performed using Trinityversion 2.0.6 (Haas et al., 2013). Trinity assembles short-read RNA-seq data intocontigs, which are likely (parts of) transcripts (Trinity genes). Low-abundancetranscripts can be assembled in two or more contigs if regions of the transcriptare not covered by any read. Based on sequence similarity, contigs are groupedtogether with the assumption that they represent isoforms derived fromthe same genetic locus. Kallisto version 0.42.1 (Bray et al., 2016) was used toquantify gene expression. Differential gene expression analysis was done withthe edgeR package version 3.10/5 (Robinson et al., 2010) using the estimatedcounts produced by Kallisto as input. Each transcript was annotated with thebest Arabidopsis hit (BLAST cutoff value of 1e-05). For this, Arabidopsis waschosen because of its well-annotated genome and because an extensive amountof functional analysis has been performed in comparison with the monocot rice(Oryza sativa).


Leeggangers et al.


http://www.bioinformatics.babraham.ac.uk/projects/fastqc/


GO Enrichment Analysis

Based on the differentially expressed genes identified in the high-temperaturecondition, the Plant GeneSet Enrichment Analysis Toolkit was used for GO en-richment. From these sets of differentially expressed genes, the genes also showingdifferential expression in the cold environment were removed for each weeklyinterval. The hypergeometric statistical test method and the Yekutieli (false dis-covery rate under dependency) multitest adjustment method settings were usedfor the analysis. The significance level and the false discovery rate were set at 0.05.

Clustering Analysis

Clustering of the transcripts with a similar expression patternwas donewiththeRpackage hclust usingPearson correlation as the distancemeasure (https://stat.ethz.ch/R-manual/R-devel/library/stats/html/hclust.html). To includeas many transcripts as possible, all transcripts were annotated with the name ofthe best Arabidopsis hit (cutoff value of 1e-05). The expression values werenormalized per gene, and then the z scores per time point were plotted. If the zscore for a gene at a time point is 1, this means that the expression value differsby 1 SD from the mean of the expression of this gene over all time points.

Identification of Potential Flowering Time Regulators

Protein sequences ofAtSOC1,AtFT,AtSEP1,AtTFL1, and all SPLswere usedfor BLASTx (cutoff of 1e-05) to identify the tulip transcripts with the highestsimilarity. All matching sequences were aligned, including the Arabidopsisgene, and the hit with the highest sequence similarity (greater than 50%) waschosen for further characterization.

Phylogenetic Analysis

For the AP1-like genes TGSQA and TGSQB, a maximum likelihood tree wasreconstructed with MEGA 5.0 (Tamura et al., 2011). The alignment was made withthe default ClustalW settings in MEGA 5.0. For the construction of the maximumlikelihood tree, theWhelan andGoldmanmodelwasused as the substitutionmodeland 500 bootstraps were generated to test the reliability of the tree. In addition, thesetting gaps/missing data treatment was changed to partial deletion with 95% asthe site coverage cutoff. These same settings were used to generate the maximumlikelihood tree of the FT/TFL1 protein sequences. Here, a cutoff of 70% for thebootstrap value was used to adjust the branches. The neighbor-joining tree of theMADS box proteins was constructed in a similar way but using the Dayhoff model.

Real-Time PCR for Expression Analysis

Real-time PCRwas performed in a total volume of 20mL containing 10mL ofiQ SYBR Green Supermix (Bio-Rad), 5 mL of each forward and reverse primer(0.05 mM; primer details are listed in Supplemental Table S2), and 5 mL of a 1:15dilution of the cDNA reaction mixture as template. Reactions were performedon the CFX Connect real-time PCR detection system (Bio-Rad) with an initial3-min denaturation at 95°C followed by 40 cycles of 95°C for 10 s and 60°C for30 s for the amplification. Final steps used for elongation were 95°C for 1 min,55°C for 10 s, and 95°C for 30 s with afterward a melt curve determination.Normalized expression levels were calculated by the DDCt method (Livak andSchmittgen, 2001) with TgACT as the reference gene. Calculations were basedon three technical replicates and two to three biological replicates.

Construction of Overexpression Lines in Arabidopsis

Twoselectedgeneswereamplified fromcDNAbyPCRwith theprimersTgTFL1(forward, 59-ATGGCAAGAGTGCTGGAGC-39, and reverse, 59-TCACTGCTCC-CACTTAACAT-39) and TgSOC1L2 (forward, 59-ATGAAGAGGGGGAAGA-CACA-39, and reverse, 59-CCATCCAATATGCAAGTCCG-39). ThePCR fragmentsof the flowering time genes were cloned in the Gateway overexpression vectorpGD625 (Immink et al., 2002), driving ectopic expression of the transgene from thecauliflower mosaic virus 35S promoter. All generated constructs were introducedintoAgrobacterium tumefaciensAGL0 and transformed intoArabidopsis Columbia-0plants using the floral dip method (Clough and Bent, 1998).

Yeast Two-Hybrid Assays

Yeast two-hybrid screenswereperformed according todeFolter and Immink(2011). All baitswere tested for autoactivation capacity prior to the screening for

potential protein-protein interactions. None of the tested baits showed auto-activation capacity. Saccharomyces cerevisiae, strains PJ69-4a and PJ69-4alphawere used in all two-hybrid analyses.

Accession Numbers

Sequences of the potential flowering time genes described in this study canbe found in GenBank (http://www.ncbi.nlm.nih.gov) under the accessionnumbers BAJ09453.1 (TGSQA), BAJ09452.1 (TGSQB), KY464928 (TgFT-like),KY464929 (TgSOC1L1), KY464930 (TgSOC1L2), KY464931 (TgSEP1), KY464932(TgTFL1), KY464933 (TgSPL1), and KY464934 (TgSPL2). The raw RNA-seq datahave been submitted to the Sequence Read Archive depository and can beobtained from http://www.ncbi.nlm.nih.gov/bioproject/327809.

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Temperature conditions and tulip morphologyafter transfer from the field to the climate cells.

Supplemental Figure S2. Morphological changes of the SAM during thevegetative-to-reproductive phase change in tulip.

Supplemental Figure S3. Phylogenetic analysis of TGSQA and TGSQB.

Supplemental Figure S4. General overview of the GO enrichment analysis.

Supplemental Figure S5. Selection of clusters (high-temperature condi-tion) with potential flowering time regulators.

Supplemental Figure S6. Expression patterns of a selection of 25 tulip genesthat have a high similarity to known flowering time genes in Arabidopsis.

Supplemental Figure S7. Phylogenetic analysis.

Supplemental Figure S8. Morphological analysis of the six different tulipcultivars.

Supplemental Figure S9. Alignment of tulip TgSOC1L2 and ArabidopsisAtSOC1 protein sequences.

Supplemental Table S1. Statistical overview of the de novo assembly byTrinity.

Supplemental Table S2. Quantitative PCR primers for the expression anal-ysis of tulip genes.

ACKNOWLEDGMENTS

We thank Gebroeders Klaver, Maliepaard Bloembollen, and Van der GulikTulpen for the plant material; Maarten Holdinga and Alex Silfhout for plantingthe bulbs in the field and Juliette Silven for help with gene cloning; Bioscience ofWageningen Plant Research for allowing use of the Carl Zeiss SV11 stereomi-croscope; and Mariana Silva Artur for suggestions on the construction ofphylogenetic trees.

Received November 21, 2016; accepted January 10, 2017; published January 19,2017.

LITERATURE CITED

Anderson N (2006) Flower Breeding and Genetics: Issues, Challenges andOpportunities for the 21st Century. Springer, The Netherlands

Andres F, Coupland G (2012) The genetic basis of flowering responses toseasonal cues. Nat Rev Genet 13: 627–639

Balasubramanian S, Weigel D (2006) Temperature induced flowering inArabidopsis thaliana. Plant Signal Behav 1: 227–228

Beijer JJ (1952) De ontwikkelingsstadia van tulp. Publication of the Labo-ratory of bulb research Lisse, The Netherlands. 92: 1–7

Bewley JD, Bradford KJ, Hilhorst HWM, Nonogaki H (2013) Seeds:Physiology of Development, Germination and Dormancy, Ed 3. Springer,New York

Blümel M, Dally N, Jung C (2015) Flowering time regulation in crops:what did we learn from Arabidopsis? Curr Opin Biotechnol 32: 121–129




https://stat.ethz.ch/R-manual/R-devel/library/stats/html/hclust.html

https://stat.ethz.ch/R-manual/R-devel/library/stats/html/hclust.html


http://www.ncbi.nlm.nih.gov

http://www.ncbi.nlm.nih.gov/bioproject/327809













Bolger AM, Lohse M, Usadel B (2014) Trimmomatic: a flexible trimmer forIllumina sequence data. Bioinformatics 30: 2114–2120

Borner R, Kampmann G, Chandler J, Gleissner R, Wisman E, Apel K,Melzer S (2000) A MADS domain gene involved in the transition toflowering in Arabidopsis. Plant J 24: 591–599

Botschantzeva Z (1982) Tulips: Taxonomy, Morphology, Cytology, Phy-togeography and Physiology. Balkema, Rotterdam, The Netherlands

Box MS, Huang BE, Domijan M, Jaeger KE, Khattak AK, Yoo SJ, SedivyEL, Jones DM, Hearn TJ, Webb AA, et al (2015) ELF3 controls ther-moresponsive growth in Arabidopsis. Curr Biol 25: 194–199

Bray NL, Pimentel H, Melsted P, Pachter L (2016) Near-optimal probabi-listic RNA-seq quantification. Nat Biotechnol 34: 525–527

Capovilla G, Pajoro A, Immink RGH, Schmid M (2015) Role of alternativepre-mRNA splicing in temperature signaling. Curr Opin Plant Biol 27:97–103

Choi K, Kim J, Hwang HJ, Kim S, Park C, Kim SY, Lee I (2011) TheFRIGIDA complex activates transcription of FLC, a strong floweringrepressor in Arabidopsis, by recruiting chromatin modification factors.Plant Cell 23: 289–303

Christenhusz MJM, Govaerts R, David JC, Hall T, Borland K, Roberts PS,Tuomisto A, Buerki S, Chase MW, Fay MF (2013) Tiptoe through thetulips: cultural history, molecular phylogenetics and classification ofTulipa (Liliaceae). Bot J Linn Soc 172: 280–328

Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16: 735–743

Compton TJ, Rijkenberg MJA, Drent J, Piersma T (2007) Thermal toler-ance ranges and climate variability: a comparison between bivalvesfrom differing climates. J Exp Mar Biol Ecol 352: 200–211

Cui Z, Zhou B, Zhang Z, Hu Z (2013) Abscisic acid promotes flowering andenhances LcAP1 expression in Litchi chinensis Sonn. S Afr J Bot 88: 76–79

de Folter S, Immink RGH (2011) Yeast protein-protein interaction assaysand screens. In L Yuan, ES Perry, eds, Plant Transcription Factors:Methods and Protocols. Humana Press, Totowa, NJ, pp 145–165

de Folter S, Immink RGH, Kieffer M, Parenicová L, Henz SR, Weigel D,Busscher M, Kooiker M, Colombo L, Kater MM, et al (2005) Com-prehensive interaction map of the Arabidopsis MADS box transcriptionfactors. Plant Cell 17: 1424–1433

De Hertogh A, Le Nard M (1993) The Physiology of Flower Bulbs: AComprehensive Treatise on the Physiology and Utilization of Orna-mental Flowering Bulbous and Tuberous Plants. Elsevier, New York

Fernández V, Takahashi Y, Le Gourrierec J, Coupland G (2016) Photo-periodic and thermosensory pathways interact through CONSTANS topromote flowering at high temperature under short days. Plant J 86:426–440

Fornara F, de Montaigu A, Coupland G (2010) SnapShot: control offlowering in Arabidopsis. Cell 141: 550.e1–550.e2

Frankowski K, Wilmowicz E, Ku�cko A, Kęsy J, �Swie _zawska B, KopcewiczJ (2014) Ethylene, auxin, and abscisic acid interactions in the control ofphotoperiodic flower induction in Pharbitis nil. Biol Plant 58: 305–310

Galvão VC, Collani S, Horrer D, Schmid M (2015) Gibberellic acid sig-naling is required for ambient temperature-mediated induction offlowering in Arabidopsis thaliana. Plant J 84: 949–962

Garciarrubio A, Legaria JP, Covarrubias AA (1997) Abscisic acid inhibitsgermination of mature Arabidopsis seeds by limiting the availability ofenergy and nutrients. Planta 203: 182–187

Gilford JM, Rees AR (1973) Growth of the tulip shoot. Sci Hortic (Amsterdam)1: 143–156

Haas BJ, Papanicolaou A, Yassour M, Grabherr M, Blood PD, Bowden J,Couger MB, Eccles D, Li B, Lieber M, et al (2013) De novo transcriptsequence reconstruction from RNA-seq using the Trinity platform forreference generation and analysis. Nat Protoc 8: 1494–1512

Hanano S, Goto K (2011) Arabidopsis TERMINAL FLOWER1 is involved inthe regulation of flowering time and inflorescence development throughtranscriptional repression. Plant Cell 23: 3172–3184

Hirai M, Ochiai T, Kanno A (2010) The expression of two DEFICIENS-likegenes was reduced in the sepaloid tepals of viridiflora tulips. Breed Sci60: 110–120

Huang NC, Jane WN, Chen J, Yu TS (2012) Arabidopsis thalianaCENTRORADIALIS homologue (ATC) acts systemically to inhibit floralinitiation in Arabidopsis. Plant J 72: 175–184

Huijser P, Schmid M (2011) The control of developmental phase transitionsin plants. Development 138: 4117–4129

Immink RGH, Gadella TWJ, Ferrario S, Busscher M, Angenent GC (2002)Analysis of MADS-box protein-protein interactions in living plant cells.Proc Natl Acad Sci USA 99: 2416–2421

Irish VF, Sussex IM (1990) Function of the apetala-1 gene during Arabi-dopsis floral development. Plant Cell 2: 741–753

Jeong S, Clark SE (2005) Photoperiod regulates flower meristem develop-ment in Arabidopsis thaliana. Genetics 169: 907–915

Jofuku KD, den Boer BG, Van Montagu M, Okamuro JK (1994) Control ofArabidopsis flower and seed development by the homeotic gene APETALA2.Plant Cell 6: 1211–1225

Kamenetsky R, Okubo H (2012) Ornamental Geophytes: From Basic Sci-ence to Sustainable Production. CRC Press, Boca Raton, FL

Kanno A, Nakada M, Akita Y, Hirai M (2007) Class B gene expression andthe modified ABC model in nongrass monocots. Sci World J 7: 268–279

Khodorova NV, Boitel-Conti M (2013) The role of temperature in thegrowth and flowering of geophytes. Plants (Basel) 2: 699–711

Komiya R, Ikegami A, Tamaki S, Yokoi S, Shimamoto K (2008) Hd3a andRFT1 are essential for flowering in rice. Development 135: 767–774

Koskela EA, Mouhu K, Albani MC, Kurokura T, Rantanen M, SargentDJ, Battey NH, Coupland G, Elomaa P, Hytönen T (2012) Mutation inTERMINAL FLOWER1 reverses the photoperiodic requirement for flower-ing in the wild strawberry Fragaria vesca. Plant Physiol 159: 1043–1054

Kumar SV, Lucyshyn D, Jaeger KE, Alos E, Alvey E, Harberd NP, WiggePA (2012) Transcription factor PIF4 controls the thermosensory activa-tion of flowering. Nature 484: 242–245

Lambrechts H, Rook F, Kolloffel C (1994) Carbohydrate status of tulipbulbs during cold-induced flower stalk elongation and flowering. PlantPhysiol 104: 515–520

Lee J, Lee I (2010) Regulation and function of SOC1, a flowering pathwayintegrator. J Exp Bot 61: 2247–2254

Lee JH, Ryu HS, Chung KS, Posé D, Kim S, Schmid M, Ahn JH (2013)Regulation of temperature-responsive flowering by MADS-box tran-scription factor repressors. Science 342: 628–632

Leeggangers HACF, Moreno-Pachon N, Gude H, Immink RGH (2013)Transfer of knowledge about flowering and vegetative propagationfrom model species to bulbous plants. Int J Dev Biol 57: 611–620

Li SF, Milliken ON, Pham H, Seyit R, Napoli R, Preston J, Koltunow AM,Parish RW (2009) The Arabidopsis MYB5 transcription factor regulatesmucilage synthesis, seed coat development, and trichome morphogen-esis. Plant Cell 21: 72–89

Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression datausing real-time quantitative PCR and the 2(-D D C(T)) method. Methods25: 402–408

McCarty DR (1995) Genetic control and integration of maturation andgermination pathways in seed development. Annu Rev Plant PhysiolPlant Mol Biol 46: 71–93

McClung CR, Lou P, Hermand V, Kim JA (2016) The importance of am-bient temperature to growth and the induction of flowering. Front PlantSci 7: 1266

Mockler TC, Yu X, Shalitin D, Parikh D, Michael TP, Liou J, Huang J,Smith Z, Alonso JM, Ecker JR, et al (2004) Regulation of flowering timein Arabidopsis by K homology domain proteins. Proc Natl Acad SciUSA 101: 12759–12764

Moreno-Pachon NM, Leeggangers HACF, Nijveen H, Severing E, Hilhorst H,Immink RGH (2016) Elucidating and mining the Tulipa and Liliumtranscriptomes. Plant Mol Biol 92: 249–261

Mutasa-Göttgens E, Hedden P (2009) Gibberellin as a factor in floral reg-ulatory networks. J Exp Bot 60: 1979–1989

Nakatsubo T, Mizutani M, Suzuki S, Hattori T, Umezawa T (2008)Characterization of Arabidopsis thaliana pinoresinol reductase, a new typeof enzyme involved in lignan biosynthesis. J Biol Chem 283: 15550–15557

Noy-Porat T, Cohen D, Mathew D, Eshel A, Kamenetsky R, FlaishmanMA (2013) Turned on by heat: differential expression of FT and LFY-likegenes in Narcissus tazetta during floral transition. J Exp Bot 64: 3273–3284

Odell JT, Nagy F, Chua NH (1985) Identification of DNA sequences re-quired for activity of the cauliflower mosaic virus 35S promoter. Nature313: 810–812

Osnato M, Castillejo C, Matías-Hernández L, Pelaz S (2012) TEMPRANILLOgenes link photoperiod and gibberellin pathways to control flowering inArabidopsis. Nat Commun 3: 808

Park DH, Somers DE, Kim YS, Choy YH, Lim HK, Soh MS, Kim HJ, KaySA, Nam HG (1999) Control of circadian rhythms and photoperiodicflowering by the Arabidopsis GIGANTEA gene. Science 285: 1579–1582


Leeggangers et al.



Persson S, Caffall KH, Freshour G, Hilley MT, Bauer S, Poindexter P, HahnMG, Mohnen D, Somerville C (2007) The Arabidopsis irregular xylem8 mu-tant is deficient in glucuronoxylan and homogalacturonan, which are es-sential for secondary cell wall integrity. Plant Cell 19: 237–255

Pose D, Verhage L, Ott F, Yant L, Mathieu J, Angenent GC, Immink RGH,Schmid M (2013) Temperature-dependent regulation of flowering byantagonistic FLM variants. Nature 503: 414–417

Preston JC, Hileman LC (2010) SQUAMOSA-PROMOTER BINDINGPROTEIN 1 initiates flowering in Antirrhinum majus through the activationof meristem identity genes. Plant J 62: 704–712

Rantanen M, Kurokura T, Jiang P, Mouhu K, Hytönen T (2015) Strawberryhomologue of terminal flower1 integrates photoperiod and temperaturesignals to inhibit flowering. Plant J 82: 163–173

Rantanen M, Kurokura T, Mouhu K, Pinho P, Tetri E, Halonen L, PalonenP, Elomaa P, Hytönen T (2014) Light quality regulates flowering inFvFT1/FvTFL1 dependent manner in the woodland strawberry Fragariavesca. Front Plant Sci 5: 271

Ratcliffe OJ, Amaya I, Vincent CA, Rothstein S, Carpenter R, Coen ES,Bradley DJ (1998) A common mechanism controls the life cycle andarchitecture of plants. Development 125: 1609–1615

Rietveld PL, Wilkinson C, Franssen HM, Balk PA, van der Plas LHW,Weisbeek PJ, Douwe de Boer A (2000) Low temperature sensing in tulip(Tulipa gesneriana L.) is mediated through an increased response toauxin. J Exp Bot 51: 587–594

Robinson MD, McCarthy DJ, Smyth GK (2010) edgeR: a Bioconductorpackage for differential expression analysis of digital gene expressiondata. Bioinformatics 26: 139–140

Shannon S, Meeks-Wagner DR (1991) A mutation in the Arabidopsis TFL1gene affects inflorescence meristem development. Plant Cell 3: 877–892

Sofo A (2016) Arabidopsis thaliana: Cultivation, Life Cycle and FunctionalGenomics. Nova Science, New York

Steward FC, Barber JT, Bleichert EF, Roca WM (1971) The behavior of shootapices of Tulipa in relation to floral induction. Dev Biol 25: 310–335

Strasser B, Alvarez MJ, Califano A, Cerdán PD (2009) A complementaryrole for ELF3 and TFL1 in the regulation of flowering time by ambienttemperature. Plant J 58: 629–640

Sundström JF, Nakayama N, Glimelius K, Irish VF (2006) Direct regulationof the floral homeotic APETALA1 gene by APETALA3 and PISTILLATAin Arabidopsis. Plant J 46: 593–600

Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011)MEGA5: molecular evolutionary genetics analysis using maximum like-lihood, evolutionary distance, and maximum parsimony methods. MolBiol Evol 28: 2731–2739

Van Dam MFN, Haaster AJM (2013) Onderzoek naar de oorzaak van vroegebloemverdroging in tulpen. Praktijkonderzoek Plant en Omgeving BBF,Lisse, The Netherlands. pp 1–29

Van der Toorn A, Zemah H, Van As H, Bendel P, Kamenetsky R (2000)Developmental changes and water status in tulip bulbs during storage:visualization by NMR imaging. J Exp Bot 51: 1277–1287

van Dijk ADJ, Morabito G, Fiers M, van Ham RCHJ, Angenent GC,Immink RGH (2010) Sequence motifs in MADS transcription factorsresponsible for specificity and diversification of protein-protein inter-action. PLOS Comput Biol 6: e1001017

Verhage L, Angenent GC, Immink RGH (2014) Research on floral timingby ambient temperature comes into blossom. Trends Plant Sci 19: 583–591

Villacorta-Martin C, Núñez de Cáceres González FF, de Haan J, HuijbenK, Passarinho P, Lugassi-Ben Hamo M, Zaccai M (2015) Whole tran-scriptome profiling of the vernalization process in Lilium longiflorum(cultivar White Heaven) bulbs. BMC Genomics 16: 550

Wahl V, Ponnu J, Schlereth A, Arrivault S, Langenecker T, Franke A, FeilR, Lunn JE, Stitt M, Schmid M (2013) Regulation of flowering bytrehalose-6-phosphate signaling in Arabidopsis thaliana. Science 339:704–707

Wang AX, Wang DY (2009) Regulation of the ALBINO3-mediated transi-tion to flowering in Arabidopsis depends on the expression of CO andGA1. Biol Plant 53: 484–492

Wang WY, Chen WS, Chen WH, Hung LS, Chang PS (2002) Influence ofabscisic acid on flowering in Phalaenopsis hybrida. Plant Physiol Biochem40: 97–100

Wang Y, Li L, Ye T, Lu Y, Chen X, Wu Y (2013) The inhibitory effect of ABAon floral transition is mediated by ABI5 in Arabidopsis. J Exp Bot 64:675–684

Yamaguchi A, Kobayashi Y, Goto K, Abe M, Araki T (2005) TWIN SISTEROF FT (TSF) acts as a floral pathway integrator redundantly with FT.Plant Cell Physiol 46: 1175–1189

Yamaguchi T, Hirano HY (2006) Function and diversification of MADS-boxgenes in rice. Sci World J 6: 1923–1932





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