ORIGINAL ARTICLE

TrMADS3, a new MADS-box gene, from a perennial speciesTaihangia rupestris (Rosaceae) is upregulated by coldand experiences seasonal fluctuation in expression level

Xiaoqiu Du & Qiying Xiao & Ran Zhao & Feng Wu &

Qijiang Xu & Kang Chong & Zheng Meng

Received: 9 September 2007 /Accepted: 2 April 2008 /Published online: 9 May 2008# Springer-Verlag 2008

Abstract In many temperate perennial plants, floral tran-sition is initiated in the first growth season but thedevelopment of flower is arrested during the winter toensure production of mature flowers in the next spring. Themolecular mechanisms of the process remain poorlyunderstood with few well-characterized regulatory genes.Here, a MADS-box gene, named as TrMADS3, was isolatedfrom the overwintering inflorescences of Taihangia rupest-ris, a temperate perennial in the rose family. Phylogeneticanalysis reveals that TrMADS3 is more closely related tothe homologs of the FLOWERING LOCUS C lineage thanto any of the other MIKC-type MADS-box lineages knownfrom Arabidopsis. The TrMADS3 transcripts are extensivelydistributed in inflorescences, roots, and leaves during thewinter. In controlled conditions, the TrMADS3 expressionlevel is upregulated by a chilling exposure for 1 to 2 weeksand remains high for a longer period of time in warmconditions after cold treatment. In situ hybridization revealsthat TrMADS3 is predominately expressed in the vegetativeand reproductive meristems. Ectopic expression ofTrMADS3 in Arabidopsis promotes seed germination on

the media containing relatively high NaCl or mannitolconcentrations. These data indicate that TrMADS3 in aperennial species might have its role in both vegetative andreproductive meristems in response to cold.

Keywords Taihangia . Overwintering .

FLOWERING LOCUS C . MADS-box . Perennial

Introduction

The seasonal growth and flowering is crucial to plantsurvival and reproductive success in temperate region. Theperception of seasonal variation and the generation ofresponses in plants ensure growing and flowering at anoptimum time (Battey 2000; Ausín et al. 2005). The abilityof plants to respond to environmental signals and hence toregulate vegetative and reproductive growth represents anadaptive mechanism for surviving even under adverseconditions (Heggie and Halliday 2005). In winter annualor biennial plants like Arabidopsis and wheat, the strategyfor coping with winter usually involves controlling theonset of floral transition (vegetative meristems are changedinto reproductive meristems) until spring. The process offloral induction is immediately followed by the develop-ment of flowers within one growing season and is sensitiveto environmental cues. One environmental cue is the coldof winter (Schmitz and Amasino 2007). Typically, in winterannuals or biennials, the competence of floral transitiondemands vernalization, a long-term (over 4–8 weeks) coldexposure during the winter (Chouard 1960). In contrast, ina number of perennials, especially those of temperateorigins, there exists a delay between floral initiation andflower development (Lona 1968; Perry 1971). Floraltransition is initiated in the first growth season but the

Dev Genes Evol (2008) 218:281–292DOI 10.1007/s00427-008-0218-z

Communicated by K. Schneitz

Electronic supplementary material The online version of this article(doi:10.1007/s00427-008-0218-z) contains supplementary material,which is available to authorized users.

X. Du :Q. Xiao :R. Zhao : F. Wu :Q. Xu :K. Chong :Z. Meng (*)Laboratory of Photosynthesis and Environmental MolecularPhysiology, Institute of Botany, Chinese Academy of Sciences,Xiangshan,Beijing 100093, Chinae-mail: zhmeng@ibcas.ac.cn

X. Du :Q. Xiao :R. ZhaoGraduate School, Chinese Academy of Sciences,Beijing 100039, China

development of flower is arrested during the winter toensure production of mature flowers in the next spring.Endogenous and environmental factors interact to regulatethe process (Perry 1971; Heide 1974; Rohde and Bhalerao2007). Many perennial plants require exposure to lowtemperatures before active shoot growth or flower develop-ment can resume in the next growing season (Lona 1968;Perry 1971). However, owing to the difficulties of geneticand molecular analysis in perennials, the molecular mecha-nisms underlying the process are unclear to date.

MADS-box genes regulate a variety of developmentalprocesses in plant (Becker and Theissen 2003; Irish and Litt2005). The expression of some MADS-box genes has morerecently been described in overwintering buds of temperateperennials (Mazzitelli et al. 2007), suggesting that MADS-box transcription factors may be one of the primaryregulatory classes of genes responsible for regulation ofthe process. In light of this, we chose to determine if thereare MADS-box genes that may be regulated in theoverwintering tissues of Taihangia.

Taihangia is a China-specific temperate perennial poly-carpic herb within the Rosaceae family and grows in thetemperate mountainous area at an altitude of 1,000 to1,200m (Yü and Li 1980). Leaf primordia are formed onthe flanks of the shoot apical meristem. Leaves are arrangedin a rosette around the very short stem. Taihangia isinflorescence flowering and its flowering process extends totwo consecutive growing seasons. During the first season,inflorescences are formed in the axils of current year'sleaves from lateral meristems (Fig. 1). They are enclosed inthe basal part of leaf petioles near the soil surface duringthe winter. The next spring, inflorescences sprout flowerswhen environmental conditions are permissive. Taihangiaplants rely on a cycle of growth cessation in the winterfollowed by growth reactivation to ensure production ofmature flowers and seed development in the next growingseason, which is different from Arabidopsis, an annual

species. It is not yet clear to which extent MADS-box genesfrom a perennial may be involved in response to the coldsignal.

In this study, we present the characterization of a novelMADS-box gene isolated from the overwintering in-florescences of Taihangia, TrMADS3 (GenBank accessionno. EF469601). The expression characterization of theTrMADS3 gene was analyzed through real-time polymerasechain reaction (PCR), reverse-transcriptase PCR (RT-PCR),and in situ hybridization experiments. Taken together withthe data from ectopic expression of TrMADS3 in Arabidopsis,the functional characterization of TrMADS3 in Taihangiawas discussed.

Materials and methods

Plant material

Taihangia plants were grown in the outdoor fields and thegreenhouse at latitude of 39° 48′ north and longitude of116° 28′ east. Total RNA samples for 3′–5′ rapid amplifi-cation of cDNA ends (RACE) were isolated from floralbuds of outdoor plants in December at an averagetemperature of 4°C. Plant tissues for in situ analyses werecollected from the plants in the greenhouse that weretreated with low temperature at 4 ± 0.7°C for 2 weeks underlong-day conditions.

Isolation of TrMADS3

Total RNA of Taihangia was extracted using Plant RNAPurification Reagent (Invitrogen, Carlsbad, CA, USA).mRNAwas isolated from total RNA using Oligotex mRNAMini Kit (QIAGEN, Hilden, Germany). cDNA was ac-quired by the 3′ RACE method (Frohman et al. 1988) usingSuperscript™ III Reverse Transcriptase (Invitrogen, Carlsbad,CA, USA). The PCR was performed using the Oligo d (T)primer in combination with a MADS-box-specific degenerateprimer. Upstream sequence overlapping with the 3′ fragmentwas isolated using the 5′ RACE system, Version 2.0(Invitrogen, Carlsbad, CA, USA) following the manufac-turer's instructions. Three independent cDNA clones weresequenced for 3′ and 5′ RACE.

Sequence analysis

MIK regions of nucleotide and predicted amino acidsequence of TrMADS3 were used for phylogenetic analysis.The multiple sequence alignment was performed usingClustal X (Thompson et al. 1997). The neighbor-joininganalysis was performed using the MEGA3.1 software(Kumar et al. 2004) with Jones–Taylor–Thornton (JTT)

Fig. 1 Morphology of the Taihangia plant in the autumn; 1 to 8 showyoung to old rosette leaves in turn. Each inflorescence is formed in theaxil of its right leaf. Bar=2 cm. The insert shows a magnification ofthe inflorescence in the box

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distances (Jones et al. 1992). The maximum-parsimonyanalysis was performed using the PAUP4.0b10 softwarewith heuristic search replicate (random stepwise sequenceaddition with tree bisection and reconnection swapping).The maximum-likelihood (ML) analysis was performedusing the PHYML2.4.3 software (Guindon and Gascuel2003) with the JTT model of evolution (four categories).Bootstrap values were derived from 1,000 replicate runs. Atotal of 84 amino acid sequences of the MADS-box geneswere obtained from National Center for BiotechnologyInformation (www.ncbi.nlm.nih.gov) according to the sam-pling of Becker and Theissen (2003) and Reeves et al.(2007). A ML phylogenetic tree for the putative relatives ofFLOWERING LOCUS C (FLC) was reconstructed using thePHYML2.4.3 with some genes from the AGL15, SQUA,AGL2, and AGL6 subfamilies. Bootstrap values were derivedfrom 1,000 replicates. Some putative FLC relatives wereobtained using the Phytome database (www.phytome.org)including: mgut4481, lesc69000, stub45998, mcry17478,lsat44890, ptrc3076, and mdom2199. Other sequences wereobtained from The Institute for Genomic Research PlantTranscript Assemblies Database (http://plantta.tigr.org).

Quantitative real-time RT-PCR

The synthesis of first-strand cDNAwas made with the samemethod as described in the isolation of TrMADS3. TheTrMADS3-specific primers were 5′-GTA TGT GTA ATATGT ACT ATC CTA-3′ and 5′-CTG CTT CTG GCA CGCACA TGC-3′ and the ACTIN-specific primers were 5′-GTACCC TCT TTC GGT GAG AAT C-3′ and 5′-CCA ATCTAC GAA GGT TAT TCT C-3′. The real-time quantifica-tion of the first strand cDNA was performed on the CorbettRotor-Gene 3000 system using the QuantiTect SYBRGreen kit (QIAGEN, Hilden, Germany) and analyzed withthe software Rotor-Gene 6. The reaction mixture (20 μL)contained 0.5 μL of cDNA, 0.3 μM of each primer, andappropriate amounts of enzymes and fluorescent dyes asrecommended by the manufacturer (QIAGEN, Hilden,Germany). For a control reaction, no template was addedto the reaction mixture, resulting in no detectable fluores-cence signal from the reaction. The PCR conditions were setas follows: initial denaturation for 15min at 95°C, followedby 45 cycles of denaturation for 30s at 94°C, annealing for30s at 56°C, and extension for 30s at 72°C. The ACTINtranscripts were detected and used as an endogenous controlto normalize TrMADS3 expression. In each experiment, thetwo standard curves method was applied for relativequantification of the endogenous control and TrMADS3cDNA copies. Each sample was performed three times toconfirm the results.

For low-temperature treatment, Taihangia plants fromthe greenhouse under long-day conditions (16 h light–8 h

dark) were transferred to a growth chamber at 4 ± 0.7°Cwith the same photoperiod as in the greenhouse and keptfor 7 or 14 days to collect leaf samples. Then Taihangiaplants were transferred back to the greenhouse for 7, 30, or60 days to collect leaf samples (1.5 ± 0.2 cm in length),respectively.

For short photoperiod treatment, Taihangia plants fromthe greenhouse in long-day conditions were transferred to agrowth chamber with the short photoperiod (8h light and16h dark) at 20 ± 0.7°C for 14 days to collect leaf samples.

For abscisic acid (ABA) treatment, exogenous ABA(100 μM) was sprayed onto Taihangia plants in thegreenhouse for seven consecutive days. The leaves wereharvested with and without ABA treatment for the analysis.

For NaCl treatment, the pots growing Taihangia plantsin the greenhouse were immersed into distilled water withNaCl in a concentration of 200 mM (the solution waschanged every 3 days) and maintained for 9 days to collectleaf samples.

For dehydration treatment, the pots growing Taihangiaplants in the greenhouse were stopped watering until theplants became wilted and then the leaf samples werecollected.

Reverse transcriptase PCR

For RT-PCR reactions, the sense primer of TrMADS3 was5′-GCT GGG AGA AGA GAA GGA CCT C-3′ and theantisense one was 5′-CTG CTT CTG GCA CGC ACATGC-3′. The ACTIN-specific primers, 5′-GTA CCC TCT TTCGGT GAG AAT C-3′ and 5′-GTG AT(GT) AC(CT) TG(CT)CCATCA GG-3′, were designed according to the TaihangiaACTIN gene sequence. The amplification conditions were:2min at 94°C, followed by a certain number of cycles of 30sat 94°C, 30s at annealing temperatures determined by theprimers used and 1min at 72°C, this was followed by a 10-minextension at 72°C. As a control, the parallel amplificationreactions for ACTIN were performed. The ACTIN PCRconditions were as described. Every RT-PCR experimentwas repeated for three times to confirm the results.

In situ hybridization

The 3′ region of TrMADS3 was amplified using the sameprimers as described in RT-PCR, introduced into thepGEM-T vector (Promega, Madison, WI, U.S.A.), andtranscribed in vitro using the digoxigenin (DIG) RNAlabeling kit (Roche, Mannheim, Germany) to synthesize thesense and antisense TrMADS3 RNA probes. Tissue fixationand hybridization were performed as described (Li et al.2005) with the following modifications. Hybridization wasperformed at 52°C and final washes were performed at 55°Cin 0.1×SSC (NaCl sodium citrate buffer).

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Vector construction and Arabidopsis transformation

We cloned the full-length cDNA sequence of TrMADS3between a CaMV35S promoter and NOS terminator inpCAMBIA 1301. Then the vector was introduced intoAgrobacterium tumefaciens strain C58. Arabidopsis thali-ana ecotype Columbia (Col) plants were transformed by thefloral dip method (Clough and Bent 1998). T1 generationseeds were sterilized and germinated on the 1/2 MS(Murashige and Skoog 1962) solid medium plates contain-ing 25mg/L hygromycin B (Roche, Mannheim, Germany)as a selective agent in the long-day conditions (16h lightand 8h dark). Seven days later, positive T1 seedlings weretransferred to soil to grow at the same photoperiod.

Northern blotting

Total RNA was extracted from the adult plants of the twohomozygous lines in T3 generation and wild type (wt),respectively, using Trizol Regent (Invitrogen, Carlsbad,CA, USA). Twelve microgram of total RNA was separatedon a 1.2% agarose gel under denaturing conditions andtransferred to a Hybond N+ nylon membrane (AmershamBiosciences, UK). The TrMADS3-specific probe templatewas made by PCR using the same primers as described inthe RT-PCR and the 3′ region was obtained. The PCRfragments were labeled using Prime-a-Gene® LabelingSystem (Promega Madison, WI, USA). Hybridization wasperformed as described (Lü et al. 2007).

Arabidopsis germination essay

Arabidopsis ecotype Columbia (wt) and the transgenicplants were cultivated at 22°C and 8h of dark–16h of light(240 μmol m−2 s−1). Siliques were harvested and after-ripened at room temperature. Seeds for germination assaywere harvested from the individual plants grown in theidentical conditions. For germination analysis under salt ordehydration stress, after-ripened seeds were sown on 1/2MS medium in concentrations ranging from 75 to 200 mMNaCl, 10 to 50 mM LiCl, and 100 to 500 mM mannitol.The seeds were first stratified at 4°C for 2 days in the darkand then transferred to normal growth conditions (22°C and16–8 light–dark cycle). For germination analysis under lowtemperature, the plates were transferred to a growthchamber at 12 ± 0.7°C or 9 ± 0.7°C. Germination wasscored every other day until 13 days after sowing. Eachgermination assay was performed with three replicates of50–100 seeds. The germination data were statisticallyanalyzed using SPSS 10.0 software. The paired t test atP = 0.05 was applied for significance in the differencebetween each transgenic line and wt.

Results

Sequence features of TrMADS3

The cDNA of a MADS-box gene was isolated by theRACE method from Taihangia and the corresponding genenamed TrMADS3. The isolated TrMADS3 cDNA is 925bplong. The sequence containing the coding region with theATG start codon, 3′ untranslated region (3′-UTR) and apoly (A) tail was obtained by 3′ RACE and the 5′-UTR by5′ RACE. The deduced protein sequence showed a 220-amino-acid product (Fig. 2a) including the conservativeMADS domain and the relatively conservative K domain,while the I region and C-terminal region are only poorlyconserved. These findings reveal that the protein encodedby TrMADS3 is a MIKCc-type MADS-domain protein.MIKCc-type MADS-box genes are divided into 13 majorclades (Becker and Theissen 2003). Using phylogeneticanalysis, we showed that TrMADS3 falls within a well-supported clade that includes FLC-like genes from someBrassicaceae plants, the sugar beet FLC homolog BvFL1(Reeves et al. 2007), and putative FLC relatives from someother core eudicot taxa (Fig. 2b and Fig. S1a–c). In allphylogenetic trees, TrMADS3 belonging to the clade waswell supported regardless of the methods used (neighborjoining, maximum parsimony, or maximum likelihood).The support value for the FLC clade was relatively high(81–100%). Furthermore, the detailed phylogenetic analysisshowed that TrMADS3 is grouped with a MADS-box genefrom apple tree (Malus x domestica belonging to theRosaceae; Fig. 2b). It is notable that alanine, not glycine,is present at position 111 in the K domain of TrMADS3 asin that of TrAG (Lü et al. 2007). The C-terminal region ofTrMADS3 shows an aspartate- and glutamate-rich stretchwith little similarity to other FLC-like proteins (Fig. 2a) andis highly hydrophilic.

TrMADS3 expression is upregulated by a periodof cold exposure

Because TrMADS3 was isolated from the overwinteringinflorescences of outdoor Taihangia plants, we initiallyperformed real-time PCR and RT-PCR to determine itsexpression in different tissues by extracting total RNA fromleaves, roots, and inflorescences. The results were scaledwith a constitutive control of ACTIN. The RT-PCR resultshowed an accumulation of the TrMADS3 transcriptsubiquitously in leaves, roots, and inflorescences (Fig. 3e).The real-time PCR showed that the TrMADS3 transcriptsaccumulated about 4.7-fold in inflorescences and about 5.1-fold in roots compared with those in leaves (Fig. 3a). Wefurther determined whether the expression level ofTrMADS3 changed during the period from late October to

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late January in Taihangia plants (aboveground parts). Thegene expression started to increase in late October andattained a maximal level by December but was at a verylow level in the summer (July; Fig. 3b). The TrMADS3transcript level of Taihangia plants (aboveground parts)outdoors at 4 ± 9°C was found to be significantly higherthan that of those growing in the greenhouse at 18 ± 3°Cduring the winter (Fig. 3b,f). In order to address whether ornot environmental factors like low temperature or short-dayphotoperiod promoted the TrMADS3 expression in outdoorTaihangia plants in the winter, we checked the TrMADS3expression level of Taihangia plants under low-temperature

treatment in controlled conditions. The plants from thegreenhouse were transferred to a growth chamber for coldtreatment and then back to the greenhouse. The expressionlevel of TrMADS3 increased up to about threefold after a 7-day cold exposure and about 4.5-fold after 14 days of coldtreatment. The expression level was kept high within30 days in the plants, which were transferred to a warmgrowth temperature from cold treatment and thereafterdecreased back to about 1.8-fold 60 days later (Fig. 3c,g).However, TrMADS3 expression level is almost unaffectedby short photoperiod, dehydration, salt, or ABA treatments(Fig. 3d).

Fig. 2 Sequence analysis ofTrMADS3. a Sequence align-ment of TrMADS3 with theFLC-like proteins includingFLC, MAF1-5, BoFLC3-2,BoFLC4-1, PMADS15, andBvFL1-v1. b Phylogeny of theputative FLC relatives withsome genes from the AGL15,SQUA, AGL2, and AGL6 sub-families. Branch numbers referto the percentage of 1,000 rep-licates that support the branchby the maximum-likelihoodanalysis. Names of species fromwhich the genes were isolatedare given before the names oraccession numbers

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TrMADS3 is expressed predominately in vegetativeand reproductive meristems

To further determine the temporary and spatial expressionpattern of TrMADS3, mRNA in situ hybridization analysiswas performed in the vegetative and different stages ofreproductive meristematic tissues of Taihangia plants aftera 2-week cold exposure at 4 ± 0.7°C. TrMADS3 was foundto be expressed strongly in the whole zone of the shootapical meristem, leaf primordia, and in the marginal area ofyoung leaves (Fig. 4a). In the axils of leaves, the TrMADS3

transcripts were present in lateral meristems that wouldform inflorescences (Fig. 4b). During the differentiation ofinflorescences, the gene is expressed in flower primordiaand young bracts (Fig. 4d). The TrMADS3 signal is strongin sepal and petal primordia as soon as they differentiatedand in floral meristem as well (Fig. 4e). Then, theTrMADS3 expression becomes restricted to the inner marginof the hypanthium, where stamen primordia are initiated, butwas not detected in the center region of floral meristem thatwould initiate carpel primordia (Fig. 4f). As the flowerdevelops, TrMADS3 is expressed in the carpel-forming

0.1

Arabidopsis t AGL15Arabidopsis t AGL18

100

Arabidopsis t AGL6Arabidopsis t AGL13

100

Arabidopsis t AGL3Nicotiana t EB425176Petunia h FBP23

37

Asparagus vDQ344499Arabidopsis t AGL9

91

Centaurea s TA2125Arabidopsis t AGL4Arabidopsis t AGL2

10067

29

40

94

Saccharum o TA35275Arabidopsis t AGL79

Lactuca s DW118226Malus d mdom2199

Arabidopsis t FUL44

8168

95

Mimulus g mgut4481Coffea c DV687858Solanum t stub45998Solanum l AW219962

100

Lycopersicon e lesc69000Petunia h PMADS15

83100

83

54

Lactuca sativa lsat44890Taraxacum o TA2622

100

31

Beta v BvFL1 v2Mesembryanthemum c mcry17478

100

36

Arabidopsis t MAF5Arabidopsis t MAF4

100

Arabidopsis t MAF1Arabidopsis t MAF3Arabidopsis t MAF2

9099

95

Brassica r BrFLCSinapis a EF542803

100

Arabidopsis t FLCThellungiella ThFLC

Brassica o BoFLC3 2Raphanus s AY273160

Brassica o BoFLC4 156

7581

7199

99

24

Populus t ptrc3076Citrus c TA6103

55

23

Vitis v TA47728Taihangia r TrMADS3

Malus d TA38584100

76

Helianthus a DY917953Lactuca s TA2396

Carthamus t TA1799Centaurea s TA634Centaurea s EH776903

96100

99100

56

99

58

49

AGL15

AGL6

AGL2

SQUA

BFig. 2 (continued)

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region, as well as developing stamens, sepals, and petals(Fig. 4g). When all four whorl organs were visible, the signalwas only detected in the developing stamens and carpels(Fig. 4h). The expression pattern study reveals thatTrMADS3 is highly expressed in those tissues that rapidlyproliferate, like the shoot apical meristem, leaf primordia,lateral meristems, and flower organ primordia.

Ectopic expression of TrMADS3 promotes seedgermination under relatively high NaCl or mannitol stressin Arabidopsis

Due to the technical difficulties in genetic transformation ofTaihangia, we constructed transgenic Arabidopsis plantsconstitutively expressing TrMADS3 under control of thecauliflower mosaic virus 35S promoter to study thefunction of TrMADS3. Two independent T3 homozygouslines (line 7 and line 16), in which the expression levels ofTrMADS3 were high according to RNA blotting (Fig. 5a),were selected for further analyses. We examined thetransgenic Arabidopsis plants under controlled conditions

of low temperature. Additionally, considering the fact thatTrMADS3 shared the property of high hydrophilicity in theC-terminal region with some cold or dehydration-respon-sive proteins (Thomashow 1998), we also examined thetransgenic Arabidopsis plants under salt or dehydrationstress. No significant differences in germination ratio wereobserved between wild-type and the 35S::TrMADS3 cDNAArabidopsis in low temperature (9°C or 12°C). However, at150 mM NaCl concentration, about 63% of wt seedsgerminated by the 11th day, while 72% (line7) and 84%(line16) of the transgenic seeds did (Fig. 5b,c). At 175-mMNaCl concentration, 47% of wt seeds compared to 63% and73% of the transgenic Arabidopsis ones germinated by the11th day (Fig. 5b). In the presence of 400 mM mannitol,two transgenic lines have 76% and 78% germinationfrequencies, respectively, by the 11th day, while wt have68% (Fig. 5d). At 450 mM mannitol, the wt germinationwas 47% and the transgenic were 72% and 81% (Fig. 5d).NaCl treatment exerts its detrimental effects on plantsbecause of ion toxicity and/or osmotic stress (Liu and Zhu1997). To determine whether the transgenic seeds are

Fig. 3 Real-time PCR and RT-PCR analysis on TrMADS3transcript levels in different tis-sues, seasons, and in response toabiotic stresses or ABA appli-cation. a Leaves, roots, andinflorescences collected fromTaihangia plants outdoors dur-ing the winter. b Taihangiaplants in the greenhouse andoutdoors in late October, No-vember, December, late January,and July. c Leaves collectedfrom Taihangia plants beforeand after cold treatment. Lengthof cold exposure is shown as“d”. LT0 samples were har-vested directly from cold-treatedplants. LT7, LT30, and LT60indicate the number of days forwhich the Taihangia plants wereback in the growth conditionsafter 14 days of cold. d Leavescollected from Taihangia plantsunder short photoperiod, dehy-dration, 200 mM NaCl, or100 μM ABA treatment. Themean values and standard devi-ations of triplicate experimentsare shown. e–g The RT-PCRsshow the TrMADS3 expressionnormalized against ACTIN. ThePCR cycle numbers are indicat-ed in parentheses

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tolerant to ion toxicity or osmotic stress, the germinationtests of the transgenic plants was conducted on mediacontaining various concentrations of LiCl because Li+ is amore toxic analog for Na+ (Mendoza et al. 1994; Wu et al.1996). As shown in Table S1, the germinating frequenciesfor each line were similar to wt under Li+ stress. Thestatistical analysis of germination data was shown inTable S1. These results indicated that the germinationfrequency of the 35S::TrMADS3 cDNA transgenic seedswas higher than wild type in the presence of relatively highconcentrations of NaCl and mannitol.

Discussion

Is the Taihangia TrMADS3 gene homologousto the Arabidopsis FLC-like genes?

FLC homologs have been so far isolated from a minority ofthe core eudicot taxa only comparing to some othersubfamilies in the MADS-box gene family. TrMADS3belongs to the clade that includes the Arabidopsis FLC-like genes (Fig. 2b; Fig S1a–c). Phylogenetic relationshipsamong FLC-like genes have been reported in several

Fig. 4 In situ mRNA localiza-tion of TrMADS3 in vegetativeand reproductive meristems.Longitudinal sections of apicesand inflorescences hybridizedwith the TrMADS3 antisenseRNA probe (the 3′ region wasused; a, b, d–h) or the sense one(c, i) are shown. a Distributionof the TrMADS3 transcriptsduring vegetative development.c The sense control of vegeta-tive meristems. b, d–h Distribu-tion of the TrMADS3 transcriptsat different stages of reproduc-tive development. i The sensecontrol of flower. (Bars=200 μm) Abbreviation: shootapical meristem (SAM), lateralmeristem (lm), bract (br), petiole(pt), floral primordium (fp), flo-ral meristem (fm), stamen pri-mordium (sp), stamen (st), sepal(se), petal (pe), carpel primordi-um (cp), and carpel (ca)

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studies (Becker and Theissen 2003; Reeves et al. 2007).The group of the FLC-like genes from Brassicaceae plantsis inferred, as it is supported by bootstrap values above99%. However, the taxa sampled in our study or otheranalyses (Becker and Theissen 2003; Reeves et al. 2007)were unbalanced with the majority of well-characterizedFLC-like genes from Brassicaceae plants. For example,PMADS15 from Petunia (Vandenbussche et al. 2003), FLChomologs from poplar (Leseberg et al. 2006), and BvFL1from sugar beet (Reeves et al. 2007) cluster with theArabidopsis FLC-like genes but form distinct outgroupswithin the FLC-like subfamily. In future studies, by addingnew FLC homologs from more taxa and conductingextensive phylogenetic analysis, the relationships amongthe members of the FLC-like subfamily will be clarified. In

some Brassicaceae plants, the FLC homologs show above75% identity to FLC in amino acid sequence (Tadege et al.2001; Schranz et al. 2002; Lin et al. 2005). Outside theplant family Brassicaceae, BvFL1 from sugar beet is 55%similar to FLC (Reeves et al. 2007). PMADS15 fromPetunia exhibits 62% similarity to FLC. TrMADS3 is only54% similar to FLC. High rates of nucleotide substitutionsduring evolution may result in low similarity among theFLC homologs from different plant families (Reeves et al.2007). This hypothesis is supported by the fact that the FLCclade in Arabidopsis is under positive Darwinian selection(Martinez-Castilla and Alvarez-Buylla 2003). In addition,TrMADS3 has tandem repetitions of single amino acid likealanine and aspartate. Single amino acid repeats (SAARs)can be functionally significant (Depledge and Dalby 2005).

Fig. 5 Germination frequencies of after-ripened seeds overexpress-ing TrMADS3 and wild-type ones under high salt or dehydrationstress. a The RNA gel-blot analysis showing the TrMADS3 transcriptlevels in the two independent 35S::TrMADS3 transgenic lines.b Germination frequencies of seeds from the two transgenic lines

and wild type on 75 to 175-mM NaCl concentrations scored at day 11after sowing. c Germination scored at day 5, 7, 9, and 12 of imbibitionon 150 mM NaCl. d 100 to 500 mM mannitol scored at day 11 aftersowing. Germination frequencies are given as the average of threeparallel tests±standard deviations

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Examples of functional SAARs have been seen in proteinswhich are associated with development and transcriptionalregulatory capacities in animal studies (Katti et al. 2000). Insome cases, SAARs can act as spacers between the domains(Golding 1999) or be useful for structural packing (Katti etal. 2000). They may also provide binding sites for protein–protein interactions (Karlin et al. 2002). The sequencediscrepancy between TrMADS3 and other well-characterizedFLC-like genes might imply their difference in function.

The expression implication of TrMADS3 in Taihangia

The FLC-like subfamily seems to be a clade of temperature-sensitive MADS-box genes. The TrMADS3 expression isupregulated in response to cold signal. FLC and some FLCorthologs from a variety of Brassicaceae plants are down-regulated by vernalization (Michaels and Amasino 1999;Sheldon et al. 1999; Tadege et al. 2001; Lin et al. 2005). ForFLC paralogs in Arabidopsis, MAF2 is responsive to briefcold spells. MAF3 and MAF4 are downregulated byvernalization, whereas MAF5 is upregulated by vernalization(Ratcliffe et al. 2003). The BvFL1 expression in sugar beet isdownregulated by vernalization but reversed after the plantsare back to the growth conditions (Reeves et al. 2007). Inmany temperate perennial species including Taihangia, thedevelopment of floral primordia that were established duringthe late summer is arrested to prevent further developmentby the process of bud dormancy in the winter (Perry 1971).TrMADS3 undergoes seasonal fluctuations in expressionlevel. The expression pattern coincides with the annualgrowth cycle of Taihangia plants, which involve alternationsbetween active growth and dormancy that are closely timedwith seasonal changes in the local climate. It is speculatedthat the FLC-like genes may be involved in dormancyregulation in perennials (Rohde and Bhalerao 2007). Thestudy in poplar showed that the expression of PtFLC, anFLC homolog from poplar, declines in dormant buds duringcold temperature exposure, indicating that it may bedifferentially regulated during dormancy release (Chen andColeman 2006). Our in situ hybridization experimentsshowed that the TrMADS3 transcripts were especiallyabundant in vegetative and reproductive meristems respon-sive to cold. Non-cold-treated Taihangia plants displayed alow level of the signal in the meristems (data not shown).Previous studies have shown that some dormancy-relatedgenes are regulated in the meristem during the process ofdormancy (Schrader et al. 2004; Rohde and Bhalerao 2007).Homeobox protein KNOTTED-1-like 2 (KNAP2) from appleis highly expressed in the shoot meristem and leafprimordia in the vegetative buds that will remain at restin the spring and is hence considered as a negative markerfor growth potential (Brunel et al. 2002). High levels ofthe AUXIN RESPONSE FACTOR 6 transcripts are detected

in the apical meristems in the potato apical tuber buds ondormancy release (Faivre-Rampant et al. 2004).

Ectopic expression of TrMADS3 does not affect flower-ing time in transgenic Arabidopsis (ecotype Columbia andLandsberg erecta, data not shown), while overexpression ofFLC causes a late-flowering phenotype in Ler (Sheldonet al. 1999; Ratcliffe et al. 2001; Scortecci et al. 2001;Ratcliffe et al. 2003). This is in line with the expression ofTrMADS3 in Taihangia, suggesting that TrMADS3 proteinmay not play a similar role as FLC in Arabidopsis. Thesugar beet FLC homolog BvFL1 functions as a repressor offlowering in transgenic Arabidopsis but does not appear tobe the bolting gene determining the presence of vernaliza-tion requirement in sugar beet (Reeves et al. 2007).Considering the importance of FLC in the integration offlowering signals in Arabidopsis (Sheldon et al. 2000;Mouradov et al. 2002; Amasino 2004), it is interesting thatthe FLC homologs outside the Brassicaceae have not beendescribed to play a role analogous to FLC yet. The FLC-like genes well characterized in some winter annuals orbiennials appear to share a conserved sequence feature justupstream of the terminal codon, potentially the C-terminusmotif, preceded by a less conservative region of variablelength. TrMADS3 is, at sequence level, not enough to beregarded as individual orthologs to any FLC-like genes inArabidopsis. The case for the group of FLC homologs inpoplar (a perennial tree) genome is similar (Leseberg et al.2006). The C-terminal region of TrMADS3 does not containthe potential motif and is less similar to those of the otherFLC-like genes. Alterations in regulatory and coding regionsequences of these homologous genes might result in theirfunctional changes during evolution.

On relatively high concentrations of mannitol and NaCl,ectopic expression of TrMADS3 promotes seed germinationof Arabidopsis. Unlike NaCl, LiCl have similar effects onthe germination of wt and transgenic plant. These resultssuggest that the transgenic plants appear primarily totolerate osmotic stress exerted by NaCl and mannitol.When plants are subjected to stress leading to cellulardehydration, a number of hydrophilic proteins like late-embryogenesis-abundant (Rorat 2006) and antifreeze pro-teins (Griffith and Yaish 2004) are induced or upregulated.The hydrophilic C-terminal of TrMADS3 implies theactivity of the protein under dehydration conditions inTaihangia. FLC homologs have not been reported as relatedto dehydration tolerance. However, a role of FLC indehydration avoidance has been proposed by the analysisof a number of genotypes of Arabidopsis (McKay et al.2003), suggesting protection against dehydration is animportant selective factor during evolution. Given thefact that Taihangia's preferred extreme habitat of clifffaces (Shen et al. 1994) and water content of over-wintering tissues decreases during the winter in temperate

290 Dev Genes Evol (2008) 218:281–292

perennials (Welling et al. 2002), the role of TrMADS3during the winter might be related to the adaptability towater deficit.

In summary, TrMADS3 is regulated by temperature andexperiences seasonal fluctuation in expression level in aperennial species. The functions of FLC homologs inperennials may be an exciting avenue for future research.At present it is difficult to identify the specific role ofTrMADS3 according to our data. Therefore, more studies onTrMADS3 by reverse genetic analyses (overexpression orsilencing of the gene in Taihangia) are necessary to deeplyunderstand the function of the gene.

Acknowledgements We thank Suzhen Zhao, Hongyan Shan, andKunmei Su for lab assistance. We are grateful to Prof. Dr. GünterTheissen for critical reading of the manuscript. We thank Dr. HongzhiKong for his helpful suggestions on phylogenetic analysis. We alsothank all anonymous reviewers for helpful comments on themanuscript. This work was supported by National Nature ScienceFoundation of China (30121003, 30770212, and 30530090).

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