evolution of the pebp gene family in plants: functional · evolution of the pebp gene family in...

Evolution of the PEBP Gene Family in Plants: FunctionalDiversification in Seed Plant Evolution1[W][OA]

Anna Karlgren, Niclas Gyllenstrand, Thomas Kallman, Jens F. Sundstrom, David Moore,Martin Lascoux, and Ulf Lagercrantz*

Department of Plant Ecology and Evolution, Uppsala University, SE–752 36 Uppsala, Sweden (A.K., T.K.,D.M., M.L., U.L.); Department of Plant Biology and Forest Genetics, Swedish University of AgriculturalSciences, SE–750 07 Uppsala, Sweden (N.G., J.F.S.); and Laboratory of Evolutionary Genomics, ChineseAcademy of Sciences-Max Planck Gesellschaft Partner Institute for Computational Biology, Chinese Academyof Sciences, 200031 Shanghai, China (M.L.)

The phosphatidyl ethanolamine-binding protein (PEBP) gene family is present in all eukaryote kingdoms, with threesubfamilies identified in angiosperms (FLOWERING LOCUS T [FT], MOTHER OF FT AND TFL1 [MFT], and TERMINALFLOWER1 [TFL1] like). In angiosperms, PEBP genes have been shown to function both as promoters and suppressors offlowering and to control plant architecture. In this study, we focus on previously uncharacterized PEBP genes fromgymnosperms. Extensive database searches suggest that gymnosperms possess only two types of PEBP genes, MFT-like and agroup that occupies an intermediate phylogenetic position between the FT-like and TFL1-like (FT/TFL1-like). Overexpressionof Picea abies PEBP genes in Arabidopsis (Arabidopsis thaliana) suggests that the FT/TFL1-like genes (PaFTL1 and PaFTL2) codefor proteins with a TFL1-like function. However, PaFTL1 and PaFTL2 also show highly divergent expression patterns. Whilethe expression of PaFTL2 is correlated with annual growth rhythm and mainly confined to needles and vegetative andreproductive buds, the expression of PaFTL1 is largely restricted to microsporophylls of male cones. The P. abies MFT-like genes(PaMFT1 and PaMFT2) show a predominant expression during embryo development, a pattern that is also found for manyMFT-like genes from angiosperms. P. abies PEBP gene expression is primarily detected in tissues undergoing physiologicalchanges related to growth arrest and dormancy. A first duplication event resulting in two families of plant PEBP genes (MFT-like and FT/TFL1-like) seems to coincide with the evolution of seed plants, in which independent control of bud and seeddormancy was required, and the second duplication resulting in the FT-like and TFL1-like clades probably coincided with theevolution of angiosperms.

The family of phosphatidyl ethanolamine-bindingproteins (PEBPs) is an evolutionarily conserved groupof proteins that occur in all taxa from bacteria to animalsand plants (Schoentgen and Jolles, 1995; Bradley et al.,1996; Banfield et al., 1998). Despite extensive sequenceconservation, PEBP genes are involved in various bio-logical processes, but the exact molecular function ofindividual genes is in most cases still unknown. Ingeneral, PEBP genes seem to act as regulators of various

signaling pathways to control growth and differentia-tion (Yeung et al., 1999; Chautard et al., 2004).

In plants, members of the PEBP gene family havebeen shown to act as key regulators of the transitionfrom the vegetative to the reproductive phase as wellas being involved in determining plant architecture(Bradley et al., 1996; Kardailsky et al., 1999; Kobayashiet al., 1999). Consequently, these members have beenintensively studied in Arabidopsis (Arabidopsis thali-ana); in particular, FLOWERING LOCUS T (FT) andTERMINAL FLOWER1 (TFL1) are among the mostthoroughly investigated PEBP proteins. Despite anamino acid identity of over 98%, these two proteinshave antagonistic functions: FT promotes flowering bymediating both photoperiod and temperature signals,while TFL1 represses it. Key amino acids responsiblefor this functional divergence have been identifiedby overexpressing chimeric versions of the two pro-teins (Hanzawa et al., 2005; Ahn et al., 2006; Pin et al.,2010). In particular, a Tyr at the position correspondingto Tyr-85 and His-99 in FT and TFL1, respectively(Hanzawa et al., 2005), and motifs in exon 4 (segmentsB and C as defined by Ahn et al. [2006]) are essen-tial for FT function. Recent analysis of a pair of FTparalogs in sugar beet (Beta vulgaris) identified threeamino acids in segment B that are important for the

1 The work was supported by the Swedish Research Council, theSwedish Research Council for Environment, Agricultural Sciences,and Spatial Planning, the Carl Tryggers Foundation, the Nilsson-Ehle Foundation, the Royal Swedish Academy of Agriculture andForestry, the European Union project TREESNIPS, the EuropeanUnion network of excellence Evoltree, and the Chinese Academy ofSciences.

* Corresponding author; e-mail [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Ulf Lagercrantz ([email protected]).

[W] The online version of this article contains Web-only data.[OA] Open Access articles can be viewed online without a sub-

scription.www.plantphysiol.org/cgi/doi/10.1104/pp.111.176206

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antagonistic effect of these genes on flowering (Pinet al., 2010). Although angiosperm FT- and TFL1-likegeneswere previously thought to be primarily involvedin the control of the switch to reproductive develop-ment, recent studies on perennials and species withsympodial growth suggest a more general role incontrolling the growth and termination of meristems(Lifschitz and Eshed, 2006; Lifschitz et al., 2006;Ruonala et al., 2008; Shalit et al., 2009).

Besides FTand TFL1, four more PEBP-like genes havebeen identified in Arabidopsis: MOTHER OF FT ANDTFL1 (MFT), BROTHER OF FTAND TFL1 (BFT), ARAB-IDOPSIS THALIANA CENTRORADIALIS (ATC), andTWIN SISTER OF FT (TSF). These genes are not as wellcharacterized, but overexpressingATC and BFT resultedin phenotypes similar to plants overexpressing TFL1,including late flowering, while overexpression of MFTresulted in a slight reduction of flowering time. How-ever, loss-of-function mutations in these genes did notdisplay any obvious phenotype (Mimida et al., 2001; Yooet al., 2004, 2010). MFTwas also reported to act duringseed germination, in agreement with its expressionpattern and that of its homologs in other plant species(Danilevskaya et al., 2008; Xi et al., 2010). The sixthmember of the gene family, TSF, is almost identical to FTat the amino acid level. Like FT, it promotes flowering,but it makes a distinct contribution only in short-dayconditions (Yamaguchi et al., 2005; D’Aloia et al., 2011).

The number of PEBP-like genes found in differentplant species varies greatly, with 19 copies identified inrice (Oryza sativa) and 24 in maize (Zea mays) comparedwith the six members found in Arabidopsis. Phyloge-netic analysis of plant PEBP genes indicates that thefamily is split into three main clades (Chardon andDamerval, 2005, and refs. therein): FT-like, TFL1-like,and a third clade that includes MFT-like genes. At-tempts to identify PEBP genes from nonangiospermplants have been more limited, but in mosses, liver-worts, and club mosses only,MFT-like orMFT-like plusa very divergent type of PEBP-like genes have beenidentified (Hedman et al., 2009). In gymnosperms, twotypes of PEBP-like genes were identified, one type sim-ilar to MFT-like genes and another type resembling FT-and TFL1-like genes (Hedman et al., 2009). In Picea abies,these two types are each represented by two copies:PaFT1 and PaFT3 belong to the MFT-like group, andPaFT2 and PaFT4 belong to the TFL1/FT-like group(Gyllenstrand et al., 2007).PaFT4 is the best characterizedof these four genes and shows an expression patternthat correlates with both photoperiod-controlled budset and temperature-mediated bud burst (Gyllenstrandet al., 2007). In general, however, the role of PEBP genesin nonangiosperm plants remains poorly known. Basedon the conclusions from this study, we renamed thesegenes as follows: the two MFT-like genes (PaFT1 andPaFT3) are subsequently referred to as PaMFT1 andPaMFT2, and the two FT/TFL1-like genes (PaFT2and PaFT4) are named PaFTL1 and PaFTL2, respectively.

The aim of this study is to extend the evolutionaryand functional studies of plant PEBP genes to non-

angiosperm plants. To elucidate the evolutionary his-tory of plant PEBP genes, we performed extensivephylogenetic inference based on available plant PEBP-like mRNA and protein sequences. This analysis sug-gests a first duplication event after the divergence ofclub mosses and seed plants, which resulted in oneMFT-like clade and one clade representing the ancestorof FT-like and TFL1-like genes. Gymnosperm FT/TFL1-like genes were placed close to the node separating FT-like and TFL1-like genes, and overexpression of PaFTL1and PaFTL2 in Arabidopsis indicates that the functionof the FT/TFL1-like proteins from gymnosperms ismore similar to the function of angiosperm TFL1-likeproteins. Based on these results, we suggest that asecond duplication of PEBP genes probably occurredin the angiosperm lineage and that the ancestral functionof the FT/TFL1-like genes was more TFL1-like. Further-more, based on expression patterns from both angio-sperms and gymnosperms, we propose that the recentlydescribed role of MFT, as a regulator of seed germina-tion, should be expanded to a more general function inembryo development.

RESULTS

Phylogenetic Reconstruction of Plant PEBP Genes

BLAST searches using PEBP-like protein sequencesfrom Arabidopsis against plant protein and nucleotidedatabases at GenBank (January 30, 2011) resulted in 265putative full-length sequences with a complete openreading frame that were kept for phylogenetic recon-structions (a complete list of accession numbers can befound in Supplemental Table S1). The general topologyof the phylogenetic tree agrees with that of Chardon andDamerval (2005) and Hedman et al. (2009), suggestingthat plant PEBP genes can be divided into three mainclusters:MFT-like, TFL1-like, and FT-like clades (Fig. 1).

Only two clusters of PEBP genes were retrieved ingymnosperms, one clearly associated with MFT-likegenes and one positioned close to the node separatingMFT-like, FT-like, and TFL1-like genes (Fig. 1). Extensiveadditional searches in available databases and tracearchives from Picea and Pinus species, representingmore than 5 million ESTs, did not reveal any additionaltype of PEBP-like genes in gymnosperms. The positionof the second gymnosperm cluster in relation to thethree major subfamilies (MFT-like, FT-like, and TFL1-like) was ambiguous. Likelihood and distance methodsplaced this cluster on the branch to theMFT-like cluster,while Bayesian methods placed this cluster with the FT-like genes in analyses based on amino acid sequencedata or with the TFL1-like cluster in analyses based onDNA sequence. However, the support for these com-peting topologies was in most cases not high (Fig. 1).

Characterization of Gymnosperm PEBP Proteins

Two MFT-like genes were identified in both sprucesand pines, and the phylogenetic analyses support a

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duplication event predating the divergence of Piceaand Pinus (Supplemental Fig. S1).Similarly, two FT/TFL1-like genes were identified in

multiple Picea species. Overall, the predicted amino acidsequences of the P. abies FT/TFL1-like genes PaFTL1 andPaFTL2 are roughly equally similar to FT and TFL1proteins in Arabidopsis. PaFTL1 shows 85% and 88%similarity to FT and TFL1, respectively, and the corre-sponding figures for PaFTL2 are 86% and 85%. Compar-ing the sequencemotifs suggested to be important for FTversus TFL1 function, PaFTL1 and PaFTL2 both containa Tyr at the position corresponding to Tyr-85 and His-88in FT and TFL1, respectively. In addition to Tyr-85/His-88 (Hanzawa et al., 2005), Ahn et al. (2006) identifiedmotifs in exon 4, in particular segments B and C, thattogether are essential for FT function. Over segments B

and C, PaFTL1 and PaFTL2 are more similar to FT thanto either TFL1 or BFT, with around 65% amino acidsidentical to FT and around 50% identical to both TFL1and BFT (Supplemental Fig. S2). However, specificamino acids in these segments that are highly conservedonly in FT homologs, and that are potentially importantfor FT function, including residues corresponding toAsp-144/Gln-140 in TFL1 and FT and a triad that isessentially conserved in FT homologs (SupplementalFig. S2; Ahn et al., 2006), were not conserved in thededuced P. abies proteins. In sugar beet, the differencebetween the two functionally antagonistic BvFT1 andBvFT2 proteins is linked to changes of three residues (Pinet al., 2010). The region containing these amino acids isdivergent between PEBP genes with activating andrepressing effects on flowering timewhen overexpressedin Arabidopsis (Supplemental Fig. S2).

PaFTL1 and PaFTL2 Genes Repress Flowering WhenOverexpressed in Arabidopsis, But No Effect on

Flowering Was Observed for PaMFT1 or PaMFT2

As a preliminary test of protein function, constructswith the four P. abies PEBP-coding sequences insertedbehind the cauliflower mosaic virus 35S promoter wereintroduced into Arabidopsis. For PaMFT1 and PaMFT2,which both belong to the MFT clade, no significantchange in flowering timewas detected (Fig. 2, A and B).Although an ANOVA indicated significant differencesbetween lines, none of the pairwise tests between thewild type and the overexpressed line were significant(Tukey’s honestly significant difference test). ForPaFTL2, however, four out of six overexpressed linesflowered significantly later than the wild type. Thesefour lines also exhibited the highest expression levelsof the transgene (Fig. 2C). Similarly, for PaFTL1, thefour lines with highest expression of the transgeneshowed significantly delayed flowering, although notas pronounced as for PaFTL2 (Fig. 2D).

In addition, the lines that expressed PaFTL2 at thehighest levels also displayed aberrant flower morphol-ogy typical for plants overexpressing TFL1 or BFT(Ratcliffe et al., 1998; Mimida et al., 2001; Yoo et al., 2010).These phenotypes included floral indeterminacy andinflorescence-in-flower-type flowers (Fig. 2, E and F).The morphology of the transgenic plant was as variableas reported for overexpressors of TFL1 (Ratcliffe et al.,1998), but the range of phenotypes observed was similar.

In conclusion, our data show that PaFTL2 can confer,at least in part, TFL1 function under overexpressionconditions. Provided that this is indicative of functionalconservation, PaFTL2 appears functionally more similarto TFL1 than to FT. Similarly, although the phenotypicdeviations observed for overexpressors of PaFTL1 wereless pronounced, the same seems to hold for PaFTL1.

P. abies PEBP Genes Have Diverged Expression Patterns

To approach the endogenous function of sprucePEBP genes, we analyzed their spatial and temporal

Figure 1. Inferred phylogeny of plant PEBP genes. Detailed treesincluding tip labels are given in Supplemental Figure S1. Supportvalues for selected branches are given for inference using Bayesian(MB), maximum likelihood (ML), and distance (NJ) methods, eachbased on DNA and amino acid sequence data. The table shows supportvalues for gymnosperm FT/TFL1-like genes attaching to branch a, b, orc. AA, Amino acids.

Evolution of the PEBP Gene Family in Plants

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expression patterns. Expression levels of PaMFT1,PaMFT2, PaFTL1, and PaFTL2 were first measured invarious tissues with quantitative reverse transcription(qRT)-PCR. RNA for these experiments was extractedfrom needles, vegetative and reproductive buds, seeds,pollen, and somatic embryos. Based on the results fromqRT-PCR, RNA in situ hybridization was then con-ducted in selected tissues.

PaFTL1 and PaFTL2

As previous data implicated PaFTL2 in the control ofannual growth rhythm (Gyllenstrand et al., 2007),expression in needles and buds was assayed in adulttrees under natural conditions at three time points ofthe growing season. PaFTL2 exhibited relatively highexpression levels in needles at all time points, whileexpression in vegetative as well as male and female

buds was high in August but low in December andMay (Fig. 3A). PaFTL1 expression was generally low inall needle and bud samples except male buds inDecember and May (Fig. 3B). PaFTL1 and PaFTL2also displayed detectable but low expression levels inpollen and seeds (Fig. 4).

Using in situ hybridization, the temporal and spatialexpression patterns of PaFTL1 were studied in malebuds, where a high expression was observed in qRT-PCR data. Expression was first seen in microsporophyllprimordia, with a seemingly even expression in entireprimordia (Fig. 5A). At later stages during autumn,expression was localized inside the microsporangia,without visible expression in tapetum cells (Fig. 5, Band C). In early spring before meiosis, expressionreached a very high level in microspore mother cells(Fig. 5, D and E). Additional estimates of PaFTL1expression later in the season using qRT-PCR showedthat expression in male bud remained high until budburst and release of pollen (data not shown). Based onqRT-PCR data, PaFTL2 displayed high expression inneedles as well as in vegetative and reproductive buds.In situ hybridization on needle tissue failed, but indormant vegetative as well as reproductive buds, ex-pression of PaFTL2 was detected primarily in the pithand procambial region. Figure 6 shows expression inmale buds, and similar patterns were also observed invegetative and female buds (data not shown).

PaMFT1 and PaMFT2

The MFT-like PaMFT1 and PaMFT2 both displayedlow expression levels in needles and buds (Fig. 3, C andD). Both PaMFT1 and PaMFT2 were highly expressedin seeds, with very high expression levels detected bothin zygotic embryo and endosperm (Fig. 4A). PaMFT1also showed high expression levels in mature pollen(Fig. 4B). Zygotic embryos are difficult to isolate atparticular stages of development, but as P. abies somaticembryos show very similar patterns of development aszygotic ones (Filonova et al., 2000), we isolated RNAfrom somatic embryos at seven developmental stages(for a description of the stages, see Supplemental Fig.S3; Larsson et al., 2008). A clear increase in expressionwas first noted, in particular for PaMFT2, from stage B(4 d after the addition of abscisic acid [ABA]), concur-rent with the occurrence of distinct embryonal mass inearly-stage embryos, and expression remained highduring embryogenesis (Fig. 4C). For PaMFT2, a declinein expression was evident in mature embryos (Fig. 4C).We also performed a detailed study of PaMFT1 andPaMFT2 expression in situ in developing somatic em-bryos. Although PaMFT2 seemed to exhibit generallyhigher expression levels than PaMFT1 in these tissues(Fig. 4C), their spatial expression patterns in embryoswere similar (Fig. 7; Supplemental Fig. S4). At the firststage that could be reliably studied, expression waslocated to the promeristematic region (Fig. 7A). Duringdevelopment of the embryos, signal was subsequentlydetected preferentially in meristematic regions, includ-

Figure 2. Phenotypes of transgenic Arabidopsis lines overexpressingP. abies PEBP genes grown under long-day conditions. A, Number ofrosette leaves at flowering of 35S::PaMFT1 lines. B, Number of rosetteleaves at flowering of 35S::PaMFT2 lines. C, Number of rosette leaves atflowering of 35S::PaFTL2 lines. D, Number of rosette leaves at flower-ing of 35S::PaFTL1 lines. E, Strong 35S::TFL1-like phenotype observedin 35S::PaFTL2 plants. F, Inflorescence-in-flower-type flower observedin 35S::PaFTL2 plants. Within each box plot, transgenic lines areordered according to expression levels of the transgene, as measured byqRT-PCR. Relative expression values are shown as dCT (CTCNTL 2CTPaFTx) relative to the wild type. White boxes depict the wild type, andgray boxes depict individual overexpression lines. Standard box plots inA to D depict median (horizontal lines) interquartile range (boxes),range (whiskers), and outliers (white circles).

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ing the root meristem, procambium, and shoot apicalmeristem (Fig. 7, B–E; Supplemental Fig. S4). At laterstages, expression was also detected in the developingprocambium of the cotelydons (Fig. 7E). Analysis ofmature zygotic embryos revealed a similar expressionpattern for both genes (Supplemental Fig. S5), butprolonged exposure also revealed expression in mostparts of the embryo (Supplemental Fig. S5, B and C).We also detected a very specific expression pattern

in one of the P. abies MFT-like genes (PaMFT1), linkedto resin ducts in male buds. In young developing malebuds, distinct signals were evident at the site whereresin ducts will form, between the procambium andthe microsporophylls (Fig. 8A). Expression was laterconfined to the epithelial cells surrounding the resinducts (Fig. 8C). No corresponding signal was detectedin vegetative or female buds or in any type of bud forthe three other P. abies PEBP genes.

DISCUSSION

Evolutionary Expansion of the Plant PEBP Gene Family

In line with previous data (Hedman et al., 2009), thephylogeny obtained in this study suggests thatMFT-likegenes are the basal clade among plant PEBP genes.MFT-like genes are present in angiosperms, gymno-sperms, lycophytes, and bryophytes, whereas theFT-like and TFL1-like clades do not include any Phys-comitrella patens or Selaginella moellendorffii PEBP genes.Furthermore, available ESTsequence data from the fernPteridium aquilinum including over 600,000 454 readsfrom gametophyte tissue contain only MFT-like PEBPgenes (Der et al., 2011). Hence, the ancestor to FT/TFL1genes started to diverge after the separation of thelycophytes and gymnosperms, likely in the commonancestor to seed plants.The second group of conifer PEBP genes (including

PaFTL1 and PaFTL2) formed a separate cluster includ-ing one sequence from Ginkgo biloba. The position of thecluster in the tree is uncertain, so based on phylogenetic

data, this second group of conifer PEBP genes cannot beclassified as FT-like or TFL1-like. So far, no additionalgymnosperm genes clustering with TFL1-like geneshave been identified in gymnosperms. Although thepresence of TFL1-like genes in conifers cannot be un-equivocally ruled out since there is no complete conifergenome available, their presence is highly unlikely.Ambitious large-scale EST sequencing projects have

Figure 3. Expression patterns of P. abies PEBPgenes in needles and vegetative and repro-ductive buds from adult trees sampled inAugust (white bars), December (light graybars), and May (dark gray bars). A, PaFTL2.B, PaFTL1. C, PaMFT1. D, PaMFT2. Relativeexpression values based on qRT-PCR areshown as dCT (CTCNTL 2 CTPaFTx). N, Needles;VB, vegetative buds; FB, female buds; MB,male buds.

Figure 4. Expression of P. abies PEBP genes in seeds, somatic embryos,and pollen. A, Relative expression of the four P. abies PEBP genes inembryo (white bars) and endosperm (gray bars). Columns are asfollows: 1, PaMFT1; 2, PaMFT2; 3, PaFTL1; 4, PaFTL2. B, Relativeexpression of the four P. abies PEBP genes in pollen. Columns are asfollows: 1, PaMFT1; 2, PaMFT2; 3, PaFTL1; 4, PaFTL2. C, Relativeexpression of PaMFT1 (white bars) and PaMFT2 (gray bars) in somaticembryos. Columns A to G refer to the stages defined in SupplementalFigure S3. Relative expression values based on qRT-PCR are shown asdCT (CTCNTL 2 CTPaFTx).





been completed in conifers, in both spruce (for Piceaglauca, 301,425 entries, and for Picea sitchensis, 175,662entries in the National Center for Biotechnology In-formation [NCBI] dbEST) and pine (for Pinus taeda,328,628 entries in NCBI dbEST). In addition, recentEST sequencing efforts using the 454 and Illuminasequencing platforms have added more than 5 mil-lion ESTs to the NCBI Sequence Read Archive, andnone of these databases contain any conifer TFL1-likegenes.

Even though the position of PaFTL1 and PaFTL2 inrelation to the major FT, TFL1, and MFT clades isuncertain, functional data indicate that the proteinsencoded by these genes possess a function moresimilar to TFL1-like genes. In particular, overexpres-sion in Arabidopsis delayed flowering rather thanpromoted it, as would be expected from a FT-likegene, and some lines where the PaFTL2 transgenewas highly expressed showed flower phenotypes typ-ical of plants overexpressing TFL1. An attractive hy-pothesis for the evolution of FT- and TFL1-like genesbased on these observations is that the duplicationand divergence between the FT-like clade and theTFL1-like clade occurred in the angiosperm lineage.The conifer FT/TFL1-like genes would then representa lineage derived from the ancestor of both thesesubfamilies. According to this hypothesis, the ances-tral function would be more similar to TFL1 thanFT, with FT function evolving in the angiospermlineage. In contrast, the alternative hypothesis, inwhich the FT and TFL1 clades diverged before theseparation of angiosperms and gymnosperms, wouldimply both a loss of TFL1-like genes in gymnosperms

and that the remaining FT-like genes acquired a TFL1-like function. Based on available data, including thefact that PaFTL2 is expressed in both needles andvegetative buds, like several TFL1-like genes in angio-sperms (Mimida et al., 2009; Mohamed et al., 2010), thefirst hypothesis seems more parsimonious.

Figure 6. In situ localization of PaFTL2 mRNA in longitudinal andtransverse sections of P. abies male cones collected after meristemtermination. A and C, Antisense probe. B and D, Sense probe. A,Transverse section showing PaFTL2 expression mainly in the pith (PH).C, Longitudinal section showing expression of PaFTL2 in the pith andprocambial region (PC). Bars = 100 mm.

Figure 5. In situ localization of PaFTL1mRNA in longitudinal sections of developing pollen cones of P. abies. A to E, Antisense probe.F, Sense probe. A, PaFTL1 is expressed early in microsporophyll primordia in buds collected in August. B, In September, PaFTL1 isexpressed in initiating pollenmother cells. C to E, PaFTL1 expression is confined to pollenmother cells throughout thewinter period atleast until late March, before meiosis. F, LateMarch sample hybridizedwith sense probe. Arrowheads point to areas of signal obtainedwith antisense probe. Bars = 100 mm.

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Expression Patterns of PaFTL1 and PaFTL2 SuggestFunctional Diversification

PaFTL1 and PaFTL2 represent a relatively recentduplication that occurred in the gymnosperm lineage.Although overexpression in Arabidopsis demon-strates that both PaFTL1 and PaFTL2 can exert similarfunctions in a heterologous system, their expressionpatterns in P. abies were clearly distinct. PaFTL2 waspredominantly expressed in needles and both vege-tative and reproductive buds, while PaFTL1 showeda more restricted expression, with high expressionconfined to male buds. The expression of PaFTL2is correlated with growth cessation and bud set, indi-cating a role in this process, as has been reportedfor FT-like genes in Populus (Bohlenius et al., 2006). InPopulus, the FT-like PtFT1 showed a reduction in ex-pression correlated with the onset of growth cessation.Furthermore, trees with artificially down-regulatedexpression of PtFT1 were more sensitive to shorteneddaylength (Bohlenius et al., 2006). In contrast, PaFTL2showed a strong induction of expression in responseto shortened daylength that correlated with the induc-

tion of growth cessation and bud set (Gyllenstrandet al., 2007), in linewith the opposite flowering responsewhen these genes were overexpressed in Arabidopsis(Bohlenius et al., 2006; this study). It is also noteworthythat PaFTL2 showed a similar expression pattern inboth vegetative and reproductive buds, suggesting thatPaFTL2, in contrast to angiosperm FT-like genes, is notprimarily involved in the induction of reproductivedevelopment. On the other hand, the expression patternof PaFTL1 indicates a role during male cone develop-ment. Expression was first detected in microsporophyllprimordia, and PaFTL1 could potentially also be in-volved in specifyingmale reproductive development. Itis notable that two FT-like genes in poplar also show apredominant expression in male reproductive organs(Poplar eFP browser; http://bar.utoronto.ca/efppop).The divergence of expression patterns between PaFTL1and PaFTL2 illustrates the prevalence of regulatorychanges in the evolution of plant PEBP genes.

FT-like as well as TFL1-like genes have been shownto possess quite variable expression patterns in differ-ent species. Some general patterns are still evident.TFL1-like genes seem to be mainly expressed in mer-

Figure 7. In situ localization of PaMFT2 mRNA in longitudinal and transverse sections of P. abies somatic embryos. A, Somaticembryo at the beginning of late embryogenesis, corresponding to Supplemental Figure S3C. Expression is located in thepromeristematic region (PR). B, Stage corresponding to Supplemental Figure S3D. Signal is evident around the root meristem region(RM) and procambium (PC). C, Stage corresponding to Supplemental Figure S3E. D and E, Stage corresponding to SupplementalFigure S3F. Signal is evident in the procambium in emerging cotelydons. F and G, Transverse sections corresponding to regions in Eas indicated with arrows. An antisense probe was used in A to C and E to G, and a sense probe was used in D. Bars = 100 mm.





istematic regions, with a preference for secondary mer-istems, including axillary meristem, vascular cambium,and intercalary meristem (Amaya et al., 1999; Zhanget al., 2005; Conti and Bradley, 2007; Danilevskayaet al., 2008, 2010; Elitzur et al., 2009). Reports ondetailed expression patterns of FT-like genes are morelimited, perhaps due to low expression levels, butexpression in leaves and apical regions includingflowers and flower organs have been reported (Arabi-dopsis eFP browser [http://www.bar.utoronto.ca/efp],Rice eFP browser [http://bar.utoronto.ca/efprice], andPoplar eFP browser [http://bar.utoronto.ca/efppop]).In contrast to TFL1-like genes, FT-like genes are oftenunder photoperiodic control, and their induction in thephloem of leaf vasculature, and subsequent transloca-tion of the protein product to the shoot apical meristem,were shown to be important for flower induction inArabidopsis and rice and probably also several otherangiosperm species (Corbesier et al., 2007; Tamaki et al.,2007; Shalit et al., 2009; Meng et al., 2011). As PaFTL2 isunder photoperiodic control in needles, it could poten-tially code for a long-distance mobile signal affectingthe growth of apical meristems. Further studies ofmRNA and protein localization of PaFTL2 are neededto answer this question.

The MFT-Like PaMFT1 and PaMFT2 Genes Are Activeduring Embryo Development

Limited information is available on the functionof MFT-like genes, but a general feature seems to bea predominant expression in seeds (Chardon and

Damerval, 2005; Danilevskaya et al., 2008; Igasakiet al., 2008). Available functional data from Arabidop-sis indicate that MFT is involved in the control ofgermination and that it may serve as a convergencepoint of ABA and GA signaling (Xi et al., 2010). TwoABA signaling components, ABI3 and ABI5, are prob-ably directly binding to the MFT promoter and regu-latingMFT transcription, ABI3 as a repressor and ABI5as an activator. Furthermore, the DELLA protein RGL2was also reported to bind the MFT promoter to en-hance expression of the gene. MFT in turn acts as arepressor of ABI5. The outcome of this complex inter-action is that MFT promotes embryo growth duringseed germination (Xi et al., 2010).

Our expression data in P. abies support a role forMFT-like genes not only in germination but also earlierduring embryo development. High expression of bothP. abies genes was detected in zygotic embryos and thesurrounding endosperm. Similarly, in P. abies somaticembryos, the expression of PaMFT1 and PaMFT2 wasdetected soon after the addition of ABA,which is crucialfor the promotion of embryo maturation (Dunstan et al.,1998; Roberts et al., 1990). ABI3 has been identified asa master regulator of maturation (Giraudat et al., 1992).As ABI3 was shown to regulate MFT, it is interestingthat a P. abies homolog to ABI3, PaVP1 (Footitt et al.,2003; Vestman et al., 2010), shows a similar temporalexpression pattern during embryo maturation as doPaMFT1 and PaMFT2. Furthermore, the expression pat-terns of PaVP1 and P. abies PEBP genes coincide inmbryos, dormant buds, as well as male strobili (Footittet al., 2003). In Populus, overexpression of PtABI3 re-sulted in the induction of both PtFT and PtMFT duringbud development induced by short days (Ruttink et al.,2007). Thus, PaVP1 might control the expression ofP. abies MFT-like genes, and possibly even other P. abiesPEBP genes, as has been reported for the homologsABI3 and MFT in Arabidopsis (Xi et al., 2010).

Expression in embryos during the maturation phasehas also been reported in maize (ZCN9, -10, and -11;Danilevskaya et al., 2008) as well as in Arabidopsis (Leet al., 2010). During this stage, the development ofembryos goes from a state of active cell division tocell enlargement, accompanied by an increase in ABAcontent. This period is also characterized by a massiveaccumulation of reserves and a decrease in watercontent. Later, during the final stage of seed develop-ment, embryos lose water and ABA and become des-iccation tolerant and metabolically inactive (Gutierrezet al., 2007; Braybrook and Harada, 2008). In line withthe observed ABA induction of MFT during germina-tion (Xi et al., 2010), MFT expression seems to followABA levels during embryo development. However, asno obvious phenotype related to embryo developmenthas been observed in mft mutants, a function of MFTduring this phase of development remains to be de-termined. It is notable that mutations in genes encod-ing most seed-specific transcription factors do notresult in a detectable phenotype (Le et al., 2010). Still,our expression data suggest a conserved function for

Figure 8. In situ localization of PaMFT1mRNA in transverse sections ofP. abies male cones. A and C, Antisense probe. B and D, Sense probe.A, PaMFT1 is expressed at sites of forming resin ducts in young malecones. C, At later stages, PaMFT1 expression is retained in epithelialcells enclosing the resin ducts. Arrowheads point to resin ducts or areaswhere those are to be formed. Bars = 100 mm.

Karlgren et al.




MFT-like genes in embryo development in gymno-sperms and angiosperms.

An Ancestral Function of Plant PEBP Genes inDormancy Regulation?

A common feature of tissues where PEBP geneexpression was detected in P. abies is preparation for“harsh conditions,” including physiological changessuch as dormancy, dehydration, and desiccation tol-erance. It is well known that bud and seed dormancyshare physiological characteristics (Wareing, 1956).During the maturation phase, embryos accumulatestorage compounds, but they also cease cell divisionand water content declines. A similar adaptation tocold and dehydration occurs when the buds enterdormancy. Likewise, pollen is a dormant organ that isgenerally tolerant to dehydration. From the observedexpression patterns of P. abies PEBP genes, it is tempt-ing to speculate that the original function of plantPEBP genes is related to growth arrest and/or dor-mancy. In line with this idea, Shalit et al. (2009) pro-posed that the tomato (Solanum lycopersicum) orthologsof FT and TFL1 (SFT and SP) regulate the balance ofdiverse growth processes rather than directly controlthe fate of cells or organs, as previously suggested.Our data also suggest that a second subfamily of

PEBP genes occurred concurrently with the evolutionof seed plants, in which bud dormancy and seeddormancy required independent control. A furtherduplication resulting in the FT and TFL1 clades, theproteins of which confer antagonistic functions, likelyoccurred with the evolution of flowering plants. It hasbeen speculated that in angiosperms, with their needfor rapid response to environmental signals, highlevels of FT function are required and that TFL1function evolved to alleviate the detrimental effectsof FT function (Shalit et al., 2009). However, the factthat FT/TFL1-like genes in P. abies seem to code forproteins with a TFL1-like function would suggest thatTFL1 function could actually be the ancestral state.

MATERIALS AND METHODS

Database Searches and Phylogenetic Reconstruction

An extensive search for plant PEBP genes was performed using Arabi-

dopsis (Arabidopsis thaliana) FT, MFT, and TFL1 protein sequences as queries in

BLASTsearches against GenBank (on January 20, 2011). A total of 265 putative

full-length plant proteins were aligned using ClustalW (Thompson et al.,

1994) and back-translated to mRNA sequences for analysis at the DNA level.

The alignments were filtered with Gblocks using default settings to remove

potentially poorly aligned positions and divergent regions (Talavera and

Castresana, 2007). Phylogenies were reconstructed by Bayesian inference

using MrBayes 3.1.2 (Huelsenbeck and Ronquist, 2001) with maximum

likelihood using Phyml 3.0 (Guindon and Gascuel, 2003) and distance

methods using MEGA 5.0 (www.megasoftware.net). In the amino acid anal-

ysis, Jones transition matrix was used, and both heterogeneity between sites

and invariable sites were allowed. In estimation of topologies from DNA data,

we used a generalized time-reversible model and estimated different rates for

each codon position. For the Bayesian analysis, two Markov runs each with

four chains heated according to default settings were performed. In each run,

every 100th tree was saved, and posterior topologies were estimated after

disregarding the first 1.5 million steps as burn in. The maximum likelihood

analyses were run with the Nearest Neighbor Interchange algorithm, and

support was estimated using an approximate likelihood-ratio test (Guindon

et al., 2010). Distance-based phylogenetic reconstruction was based on neighbor-

joining and LogDet distance for DNA data and on Jones transition matrix for

amino acid data. Distance estimates were based on the first two codon

positions, and bootstrap support was estimated from 500 runs. Trees were

visualized with FigTree (tree.bio.ed.ac.uk/software/figtree).

Isolation of Picea abies PEBP Genes

Searches against Pinus taeda, Picea glauca, and Picea sitchensis EST databases

revealed four unique putative PEBP genes in conifers. Partial or complete

cDNAs from P. abies were obtained by RT-PCR using the Onestep RT-PCR kit

(Qiagen). Template RNA for RT-PCR was extracted from male or vegetative

buds using a slightly modified extraction method described by Azevedo et al.

(2003). Partial cDNAs were extended by RACE reactions using the SMART

RACE cDNA Amplification kit (Clontech) to identify the complete coding

frame of the gene. In addition, the genomic sequence of each locus was

determined by sequencing of PCR products.

Sequences were base called with PHRED, assembled with PHRAP, and

visualized and edited in CONSED (Ewing et al., 1998; Ewing and Green,

1998; Gordon et al., 1998). The exon-intron structure for each P. abies PEPB

homolog was determined by alignment of cDNA sequences to the corre-

sponding genomic sequence (Supplemental Fig. S6).

P. abies Plant Material

Female, male, and vegetative buds, needles, and cones were collected from

adult trees of P. abies (more than 30 years old) growing at latitude 59�53#N(Uppsala, Sweden). Buds were collected on the first date they, after dissection,

could be visually determined as either female, male, or vegetative. Embryonic

cell line A.95.88.22 (Egertsdotter and von Arnold, 1993) was cultured as

described by Filonova et al. (2000).

Quantitative Gene Expression Analysis

For P. abies, total RNA was isolated from needles or buds following the

protocol described by Azevedo et al. (2003) with minor modifications, while

for Arabidopsis, the Qiagen RNeasy Mini kit was used in accordance with the

manufacturer’s recommendations. cDNAs were synthesized from 0.5 mg of

total RNA using SuperScript III reverse transcriptase and random hexamer

primers. qRT-PCR was performed according to Gyllenstrand et al. (2007) or on

a MyiQ Real-Time PCR Detection system (Bio-Rad) with the thermal condi-

tions 95�C for 7 min followed by 40 cycles of 95�C for 10 s and 60�C for 30 s.

Each reaction was performed in duplicate containing 12 mL of DyNAmo Flash

SYBRGreen (DyNAmo Flash SYBRGreen qPCR kit; Finnzymes), 0.5 mM of

each primer, and 4.75 mL of cDNA (diluted 1:100) in a total volume of 23.75 mL.

Primers used can be found in Gyllenstrand et al. (2007) except for PaFTL1,

where the primers for the MyiQ machine were as follows: forward,

5#-CCGCTACAACTTCAGCCTCT-3#; reverse, 5#-CCCATCTGCTTGAACAA-

CAC-3#. Polyubiquitin was used to normalize data. Expression values were

calculated as delta cycle threshold (dCT) values (CTcontrol – CTtarget).

In Situ Hybridization

Sense and antisense RNA probes were synthesized with in vitro transcrip-

tion using the DIG RNA Labeling kit (SP6/T7; Roche Applied Science)

according to the manufacturer’s recommendations and digested to 150-bp

fragments according to Jackson (1991). Templates for in vitro transcription

were PCR products generated from plasmids containing the coding sequences

of PaMFT1, PaMFT2, PaFTL1, and PaFTL2. Primers used to generate these

products are listed in Supplemental Table S2. In situ hybridization was

performed according to Karlgren et al. (2009).

Arabidopsis Transformation and Growth Conditions

The complete cDNA of PaMFT1, PaMFT2, PaFTL1, and PaFTL2 was

amplified using gene-specific primers with attB sites (Supplemental Table

S3). The PCR products were cloned into the pDONR 221 vector (Invitrogen).

The destination vector pMDC32 (Curtis and Grossniklaus, 2003) was used to





construct 35S:PaMFT1, 35S:PaMFT2, 35S:PaFTL1, and 35S:PaFTL2 plasmids.

The constructs were transformed using the freeze-thaw method into Agro-

bacterium tumefaciens GV3101 pMP90. A. tumefaciens-mediated transforma-

tions of Arabidopsis (Columbia) plants were performed using the floral dip

method (Clough and Bent, 1998), and homozygotic transgenic lines were

established.

Seeds from homozygous transgenic lines and the wild type were planted

on soil in a randomized manner with 24 pots in each tray. The seedlings were

grown under approximately 150 mmol m22 s21 cool-white fluorescent light in

16 h of light/8 h of dark and 22�C (long days). Flowering time was measured

by counting the final number of rosette leaves. All experiments were repeated

twice.

Sequence data from this article can be found in the GenBank/EMBL data

libraries under accession numbers JN039331 to JN039333.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1.Detailed phylogenetic trees of plant PEBP genes.

Supplemental Figure S2. Alignment of deduced amino acid sequences of

segments B and C in exon 4 (Ahn et al., 2006) of selected plant PEBP

genes.

Supplemental Figure S3. Stages of somatic embryogenesis used for qRT-

PCR and in situ hybridization experiments.

Supplemental Figure S4. In situ localization of PaMFT1 mRNA in longi-

tudinal sections of P. abies somatic embryos.

Supplemental Figure S5. In situ localization of PaMFT1 and PaMFT2

mRNA in longitudinal sections of zygotic embryos of P. abies.

Supplemental Figure S6. Inferred exon-intron structure of P. abies PEBP

genes.

Supplemental Table S1. List of genes included in phylogenetic analyses.

Supplemental Table S2. Primers used to generate probes for in situ

hybridization.

Supplemental Table S3. Primers used to clone cDNA of P. abies PEBP

genes.

ACKNOWLEDGMENTS

We are grateful to Emma Larsson for preparation, collection, and photo-

graphs of somatic embryos, to Kerstin Santesson for laboratory assistance,

and to Karl Holm for comments on earlier versions of the manuscript.

Received March 14, 2011; accepted May 30, 2011; published June 3, 2011.

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