Molecular Phylogenetics and Evolution 73 (2014) 129–139

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Molecular Phylogenetics and Evolution

journal homepage: www.elsevier .com/ locate /ympev

Differential SPL gene expression patterns reveal candidate genesunderlying flowering time and architectural differences in Mimulusand Arabidopsis

http://dx.doi.org/10.1016/j.ympev.2014.01.0291055-7903/� 2014 Elsevier Inc. All rights reserved.

Abbreviations: AM, axillary meristem; FT, FLOWERING LOCUS T; FUL, FRUITFULL;IM767, Iron Mountain Oregon population 767; LFY, LEAFY; miRNA, microRNA; PR,coastal Point Reyes California population; SBP, SQUAMOSA-PROMOTER BINDINGPROTEIN; SOC1, SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1; SPL, SQUAMOSA-PROMOTER BINDING PROTEIN LIKE; SAM, shoot apical meristem.⇑ Corresponding author. Address: 111 Jeffords Hall, 63 Carrigan Drive, Burlington,

VT 05405, USA. Fax: +1 802 656 0440.E-mail address: Jill.Preston@uvm.edu (J.C. Preston).

Stacy A. Jorgensen, Jill C. Preston ⇑Department of Plant Biology, The University of Vermont, 63 Carrigan Drive, Burlington, VT 05405, USA

a r t i c l e i n f o

Article history:Received 13 September 2013Revised 20 January 2014Accepted 28 January 2014Available online 7 February 2014

Keywords:ArabidopsisBranchingFlowering timeGrowth habitMimulus guttatusSPL genes

a b s t r a c t

Evolutionary transitions in growth habit and flowering time responses to variable environmental signalshave occurred multiple times independently across angiosperms and have major impacts on plant fitness.Proteins in the SPL family of transcription factors collectively regulate flowering time genes that havebeen implicated in interspecific shifts in annuality/perenniality. However, their potential importance inthe evolution of angiosperm growth habit has not been extensively investigated. Here we identify ortho-logs representative of the major SPL gene clades in annual Arabidopsis thaliana and Mimulus guttatusIM767, and perennial A. lyrata and M. guttatus PR, and characterize their expression. Spatio-temporalexpression patterns are complex across both diverse tissues of the same taxa and comparable tissuesof different taxa, consistent with genic sub- or neo-functionalization. However, our data are consistentwith a general role for several SPL genes in the promotion of juvenile to adult phase change and/or flow-ering time in Mimulus and Arabidopsis. Furthermore, several candidate genes were identified for futurestudy whose differential expression correlates with growth habit and architectural variation in annualversus perennial taxa.

� 2014 Elsevier Inc. All rights reserved.

1. Introduction

Evolutionary shifts in growth habit and life history traits haveoccurred multiple times independently across angiosperms, andhave been linked to geographic variation in temperature seasonal-ity, drought, and edaphic conditions (Quiros and Bauchan, 1988;Clauss and Koch, 2006; Hall and Willis, 2006; Wang et al.,2009a,b; Razafimandimbison et al., 2012). For example, Arabidopsisthaliana (Brassicaceae, rosid II) is an early-flowering monocarpicannual that displays apical dominance (Fig. 1a). Pre-reproductiveshoot growth in A. thaliana is confined to the shoot apical meristem(SAM), which eventually transitions into a racemose inflorescencebearing flowers and secondary coinflorescences (Dun et al., 2006;Ehrenreich et al., 2009; Finlayson et al., 2010) (Fig. 1a). In contrast,

the relatively late flowering polycarpic perennials Arabidopsis lyra-ta (Fig. 1b) and Arabis alpina (Brassicaceae) develop branched api-cal racemes, followed by axillary meristem (AM) outgrowth toform additional axillary inflorescences or axillary rhizomes forcontinued vegetative growth (Clauss and Koch, 2006; Wanget al., 2009a; pers. obs.).

The distantly related Mimulus guttatus species complex (Phrym-aceae, asterid I) includes both annual and perennial populations(Hall and Willis, 2006; van Kleunen, 2007). Inland annual popula-tions such as Iron Mountain 767 (IM767) flower early, developracemose inflorescences from both SAMs and AMs, and senesceafter flowering (Robinson, 1986; Baker et al., 2012; pers. obs.)(Fig. 1c). Unlike IM767, coastal perennial populations such as PointReyes (PR) flower relatively late, only develop an inflorescencefrom the SAM, and produce vegetative branches in the axils ofthe second and third leaf pairs that can develop as stolons (pers.obs.) (Fig. 1d). A recent study suggests that variation in MORE AX-ILLARY GROWTH (MAX) underlies differences in axillary branch out-growth between annual inland and perennial coastal dune M.guttatus populations (Baker et al., 2012). However, the genetic ba-sis of flowering time and meristem determinacy differences in gen-era such as Arabidopsis and Mimulus are only starting to be

Fig. 1. Inflorescence and branching architecture of annual and perennial Arabidopsis and Mimulus. (a) Vegetative A. thaliana plants form a basal rosette (light green leaves)from the shoot apical meristem (SAM). Following the transition to reproductive development, the SAM converts into an indeterminate apical inflorescence meristem (InfSAM), which gives rise to cauline leaves (dark green) and single flowers. Secondary axillary inflorescences (SAIs) develop in the axils of a few cauline leaves. The inflorescenceSAM and SAIs constitute an indeterminate raceme. (b) A. lyrata plants develop in a similar manner to A. thaliana. However, following the development of an apicalindeterminate raceme, meristems in the axils of rosette leaves expand as primary axillary inflorescences (PAIs) or rarely as vegetative axillary branches (VABs), the latter ofwhich form rhizomes. (c) Vegetative M. guttatus IM767 plants produce two pairs of leaves from the SAM and then quickly transition to reproductive development. The newlyformed inflorescence SAM gives rise to an indeterminate raceme, developing solitary flowers in the axils of leafy bracts acropetally from leaf pair three. Following apicalraceme development, axillary meristems (AMs) grow out to first form single-flowered PAIs at node two (2) and then multi-flowered PAIs at node one (1). (d) M. guttatus PRdevelops an apical indeterminate raceme at the four-leaf pair stage. However, rather than producing PAIs, AMs at more basal nodes remain unbranched at node one orelongate to form VABs at nodes two and three (3), the latter forming stolons.

130 S.A. Jorgensen, J.C. Preston / Molecular Phylogenetics and Evolution 73 (2014) 129–139

characterized (e.g. Caicedo et al., 2004; Stinchcombe et al., 2004;Shindo et al., 2006; Friedman and Willis, 2013).

It is hypothesized that changes in the regulation of SQUAMOSA-PROMOTER BINDING PROTEIN-like (SPL or SBP-box) genes havebeen important for the evolution of angiosperm growth habitand life history traits (Preston and Hileman, 2013). SPL genes com-prise a family of transcription factors that function in a diversity ofplant developmental processes (reviewed in Preston and Hileman(2013)). Functional studies, primarily in the angiosperms A. thali-ana, rice (Oryza sativa, Poaceae), and maize (Zea mays, Poaceae),and the moss Physcomitrella patens (Funariaceae), suggest thatthe most common SPL gene function is in the positive regulationof developmental transitions from juvenile to adult growth (vege-tative phase change) and vegetative to reproductive growth (repro-ductive phase change) (Wu and Poethig, 2006; Schwarz et al.,2008; Usami et al., 2009; Wang et al., 2009b; Yamaguchi et al.,2009; Preston and Hileman, 2010). However, SPL genes have alsobeen implicated in the control of branching (Jiao et al., 2010; Miuraet al., 2010; Preston and Hileman, 2010), fruit and trichome devel-opment (Unte et al., 2003; Wang et al., 2005; Manning et al., 2006;

Yu et al., 2010; Preston et al., 2012; Wang et al., 2012), sporogen-esis (Unte et al., 2003), anthocyanin accumulation (Guo et al.,2011), copper homeostasis (Nagae et al., 2008; Yamasaki et al.,2009), embryogenesis (Nodine and Bartel, 2010), leaf and liguledevelopment (Moreno et al., 1997; Lee et al., 2007; Schwarzet al., 2008; Shikata et al., 2009; Martin et al., 2010a,b), and fungaltoxin sensitivity (Stone et al., 2005). Thus, understanding the func-tional diversification of this gene family across plants will likelyhave important implications for our understanding of both plantdevelopment and its evolution.

The majority of SPL genes characterized to date are negativelyregulated by the microRNA miR156 during early vegetative devel-opment (Cardon et al., 1997, 1999; Rhoades et al., 2002; Araziet al., 2005; Guo et al., 2008; Salinas et al., 2012). In A. thaliana,maize, hybrid poplar (Populus � canadensis), and several woodyperennials, miR156 transcripts are expressed at high levels duringthe seedling stage and promote both juvenility and axillarybranching (Wang et al., 2011; Wei et al., 2012). This is the develop-mental stage at which plants are unable to respond to inductiveflowering signals (Poethig, 1990; Telfer et al., 1997; Baürle and

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Dean, 2006). However, as these plants get older, miR156 levels de-crease in leaves and other organs, probably in an age-dependentmanner (Wu and Poethig, 2006; Wang et al., 2009b; Yamaguchiet al., 2009; Lee et al., 2012; Yang et al., 2011). The down-regula-tion of miR156 causes a concomitant up-regulation of several SPLgenes, resulting in morphological and physiological changes thatmark the transition to the adult and then reproductive phase(Wu and Poethig, 2006; Gandikota et al., 2007; Wang et al.,2009b; Wu et al., 2009; Yamaguchi et al., 2009). In contrast, in P.patens, miR156 actually promotes developmental phase change,suggesting functional diversification between seed plants andmosses (Cho et al., 2012).

In addition to negative regulation through the miR156 age-dependent pathway, at least some A. thaliana SPL genes are posi-tively regulated in response to long-day photoperiods, gibberellicacid signaling, and ambient temperatures (Zhang et al., 2007; Junget al., 2012; Kim et al., 2012; Porri et al., 2012; Yu et al., 2012). Allof these pathways have long been known to induce flowering incompetent adult plants through the positive regulation of floralintegrator genes such as FT, SUPPRESSOR OF OVEREXPRESSION OFCONSTANS 1 (SOC1), FRUITFULL (FUL), and LEAFY (LFY); it is nowknown that these are direct and/or indirect targets of several SPLproteins (Klein et al., 1996; Corbesier and Coupland, 2006;Corbesier et al., 2007; Kim et al., 2012; Wang et al., 2009b;Yamaguchi et al., 2009; Preston and Hileman, 2013).

Despite recent insights into their developmental roles, func-tional redundancy between closely related paralogs has madecharacterization of A. thaliana SPL genes tricky. For example, muta-tions in the most extensively studied clade-VI gene AtSPL3 have noobvious effect on phenotype (Cardon et al., 1999). However, consti-tutive expression of miR156-resistant forms of AtSPL3, and closelyrelated AtSPL4 and AtSPL5, leads to the early production of adultcharacteristics and precocious flowering (reviewed in Huijser andSchmid (2011)). In combination with expression and DNA-bindingdata, these results strongly suggest that AtSPL3, AtSPL4, and AtSPL5are positive regulators of flowering (Wu and Poethig, 2006; Wanget al., 2009b; Yamaguchi et al., 2009). A similar role has been as-signed to AtSPL9 and AtSPL15, although functional data suggest thatAtSPL14 is actually a negative regulator of flowering (Stone et al.,2005; Schwarz et al., 2008; Usami et al., 2009).

Here we use comparative expression analyses to determine thepotential role of SPL gene family diversification in flowering time,growth habit, and their evolution in annual and perennial Arabid-opsis and M. guttatus. We test predictions of three hypotheses forArabidopsis and M. guttatus: (1) differential sub-functionalizationof SPL genes has occurred following both gene duplication and spe-ciation, (2) SPL genes function predominantly in the promotion ofjuvenile to adult phase change and flowering time, and (3) spa-tio-temporal changes in SPL gene regulation underlie variation ingrowth habit and life history traits. Despite evidence for extensivesub- or neo-functionalization within and between clades, our dataare consistent with a general role for SPL genes in the promotion offlowering, and in some cases vegetative phase change. Results ofour study also reveal candidate genes whose differential regulationmight have been important for the evolution of growth habit andbranching architecture in our focal taxa.

2. Materials and methods

2.1. Plant growth conditions

Seed of Arabidopsis thaliana (Columbia-0, CS37008), A. lyratassp. lyrata (CS22696), Mimulus guttatus IM767, and M. guttatus PRwere obtained from the Arabidopsis Biological Resource Center(The Ohio State University) and John Kelly (The University of

Kansas), respectively, and planted in Fafard #2 growing mix atthe University of Vermont greenhouse facility. To control for devel-opmental stage, seedlings that germinated on the same day weretransplanted to individual pots, and grown under long day condi-tions at 20 �C until flowering. Days to flowering and leaf numberat flowering were recorded at bolting (Arabidopsis) or when thefirst floral meristem was visible (M. guttatus), and significant differ-ences between taxa were determined using the analysis of variance(aov) function in R version 3.0.0.

2.2. Tissue collection

To determine temporal and spatial differences in expression be-tween IM767 and PR, and A. thaliana and A. lyrata, a time series ofdevelopmentally equivalent post-cotyledonary leaves and SAMswere harvested. In the case of M. guttatus, the top (youngest) leafpair was harvested from five individuals with one, two and three(flowering) IM767, or one, two, three, and four (flowering) PR leafpairs, following emergence of each new (approximately 1 cm long)leaf pair. At leaf pair one and three (IM767) or one and four (PR)vegetative and inflorescence SAMs were also harvested from fiveindividuals, respectively. In the case of A. thaliana and A. lyrata,material was harvested from similarly sized upper (youngest) ro-sette leaves and corresponding SAMs for four individuals at twoor three time points: 19 days post-germination (4 leaf stage forboth), 30 days post-germination (12 leaf stage for both), and80 days post-germination (38 leaf stage, A. lyrata only).

In addition to leaves and SAMs, stems from five individualswere dissected to capture as yet unexpanded AMs at nodes one(IM767 and PR), two (PR) and four (A. thaliana and A. lyrata) follow-ing emergence of the last leaf (Mimulus) or rosette leaf (Arabidop-sis) before flowering. The fate of each AM was predicted based oninspection of several plants per taxon following the transition toflowering: AMs at node one of IM767 and node four of A. lyrata pro-duce multi-flowered axillary inflorescences (racemes), at node oneof PR and node four of A. thaliana remain unexpanded, and at nodetwo of PR form vegetative axillary branches (Fig. 1).

2.3. RNA extraction, cDNA synthesis, and primer design

The fully sequenced genomes of A. thaliana and M. guttatus pop-ulation IM62 have 16 and 19 SPL genes, respectively, that have pre-viously been separated into eight of nine distinct clades (I–VIII)(Cardon et al., 1999; Preston and Hileman, 2013). To design quan-titative reverse transcriptase (q-RT)-PCR primers that would ex-actly match SPL genes from annual and perennial Arabidopsis andMimulus we first designed primers in Primer3 v0.4.0 (Rozen andSkaletsky, 2011) to amplify approximately 150 bp of each A. thali-ana SPL ortholog in A. lyrata, and each IM62 ortholog in IM767 andPR. RNA was extracted from pooled seedlings and leaves of flower-ing plants for each taxon using Tri-Reagent (Life Technologies)according to the manufacturer’s instructions. Following a DNasetreatment with TURBO DNA-free DNase (Life Technologies), cDNAwas synthesized using 1 lg of RNA in an iScript cDNA synthesisreaction (BioRad) according to the manufacturer’s instructions.cDNA was diluted 1:10 and 2 lL was used for standard PCR ampli-fication for each primer set. Amplicons were cloned into the pGEM-T vector (Promega) and four clones were sequenced per targetusing T7 primers at the High Throughput Genomics Center (Uni-versity of Washington).

Consensus sequences for each set of four clones were generatedand aligned with other available SPL genes (Preston and Hileman,2013) in MacClade (Maddison and Maddison, 2003). To verifyorthology with A. thaliana and M. guttatus IM62 genes in Phyto-zome (www.phytozome.net), a maximum likelihood phylogeneticanalysis was run on a nucleotide alignment of SPL genes based

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on approximately 237 bp of the conserved SBP-box domain inGARLI 2.0 (Zwickl, 2006) after model optimization in MrModelTest(Nylander, 2004) (Fig. S1). Five hundred maximum likelihood boot-strap replicates were conducted in GARLI 2.0 (Zwickl, 2006) todetermine support for each branch.

Q-RT-PCR primers were designed in Primer3 that exactlymatched a representative subset of SPL orthologs in both A. thalianaand A. lyrata or IM767 and PR (Table S1). In the case of very re-cently duplicated M. guttatus genes, showing little sequence diver-sity in IM62, primers were designed to amplify both copiessimultaneously from IM767 and PR. While resources precludedanalysis of all SPL genes, our final sampling scheme targeted 14out of 19 M. guttatus genes, and 11 out of 16 Arabidopsis genes rep-resenting SPL clades I–VIII, and allowing direct comparative analy-sis of orthologous or co-orthologous gene expression.

2.4. Gene expression

All q-RT-PCR analyses were conducted on a StepOne real-timePCR machine (Life Technologies) using 2 lL 1:10 diluted cDNAand Fast SYBR green master mix (Life Technologies) at 60 �C with40 cycles according to the manufacturer’s instructions. The effi-ciency of each primer pair was determined by comparing the num-ber of cycles at a given threshold (cT) for four dilutions of a leafcDNA template in triplicate as previously described (Scovilleet al., 2011). In cases where multiple peaks were identified, orwhere the peak melting temperature matched a primer/dimerpeak in the negative control, primers were discarded and new onesdesigned. Following primer optimization, relative expression ofeach target SPL gene was determined for three to five biologicalreplicates, with three technical replicates and a negative control,using the DCT method (Scoville et al., 2011). Briefly, A. thalianaand A. lyrata expression data were normalized using the geometricmean of two previously characterized housekeeping genesEUKARYOTIC TRANSLATION FACTOR 1 ALPHA (EF1alpha,At5g60390) and PROTEIN PHOSPHATASE 2A SUBUNIT A3 (PP2A,At1g13320) (Czechowksi et al., 2005), and each value was cor-rected for primer efficiency. The same procedure was used for nor-malization of IM767 and PR expression data using the geometricmean of the housekeeping genes EF1alpha and UBIQUITIN 5(UBQ5) (Scoville et al., 2011).

3. Results

3.1. SPL orthologs in Mimulus and Arabidopsis

In addition to the 10 SPL genes previously described in M. gutt-atus IM62 (Preston and Hileman, 2013), BLAST searches in Gen-bank and Phytozome revealed one additional M. guttatus SPLgene (MgSPL10) in clade II and eight SPL genes derived from veryrecent duplications of MgSPL1 to MgSPL5, MgSPL9, MgSBP1, andMgSBP2 (Fig. 2). A single ortholog of each of the 16 A. thalianaSPL genes was also identified for A. lyrata. Primers based on thepreviously identified M. guttatus IM62 SPL genes successfullyamplified putative orthologs from IM767 and PR. A maximumlikelihood analysis using the GTR + I + C model of evolution con-firmed orthology of the A. thaliana and A. lyrata genes, and theM. guttatus IM62, IM767, and PR genes (Fig. 2). The tree topologywas generally congruent with previous analyses in terms ofresolving nine major clades, eight of which contain Arabidopsisand/or Mimulus genes (Fig. 2) (Preston and Hileman, 2013). How-ever, similar to previous studies (Salinas et al., 2012; Preston andHileman, 2013), there was little support for between-clade rela-tionships, probably due to limited characters within the alignableSBP-box domain.

3.2. SPL gene expression during leaf development

Although both germinated quickly after a few days, days toflowering and leaf pair number at flowering were significantly dif-ferent (p < 0.001 and <0.001, respectively) between M. guttatusIM767 and PR. IM767 plants flowered on average 21.7 days(SD = 1.1) after germination with an average of 2.98 leaf pairs(SD = 0.1), whereas PR plants flowered on average 33.7 days(SD = 2.7) after germination with an average of 4.08 leaf pairs(SD = 0.3). Consistent with a role in the induction of flowering,MgSPL4, MgSBP1, MgSBP2, and MgSPL7 expression increased in adevelopmental series of leaves in both IM767 and PR (Fig. 3a). Inthe case of MgSPL4 and MgSBP1 transcript levels were at least1.5-fold higher in IM767 compared to PR at the third leaf pairstage. However, MgSBP2 and MgSPL7 expression was respectivelyhigher or similar in PR versus IM767 by leaf pair 3, and increasedeven more in leaf pair 4 of PR (Fig. 3a). A similar up-regulation ofexpression was observed for MgSPL5 and MgSPL8 in IM767, butnot PR.

A number of SPL genes were expressed in a manner consistentwith promotion of juvenile to adult phase change, which likely oc-curs by leaf pair one stage in M. guttatus. Interestingly, all of thesegenes – MgSPL1, MgSPL2, and MgSPL8 – were in the later floweringPR background, and were expressed at least 1.5-fold higher in PRleaf pair one versus all IM767 leaf pairs. In IM767, MgSPL1 andMgSPL2 showed no consistent pattern over developmental time(Fig. 3a).

Arabidopsis thaliana and A. lyrata also germinated quickly after afew days. However, under inductive long day conditions, A. lyratabolted significantly later after an average of 80 days with 38 ro-sette leaves (SD = 3.1), compared with 30 days and 12 rosetteleaves (SD = 0.8) in A. thaliana (p < 0.001 and <0.001, respectively).To determine if SPL gene expression positively correlated withdevelopmental age, gene expression was determined in leaves ofA. thaliana and A. lyrata at the same chronological and develop-mental age. Consistent with a positive role in the floral transition,transcript levels for one A. thaliana gene (SPL7) and several A. lyratagenes (SPL12, SPL14, SPL8, SPL6, SPL2, and SPL4) increased in leavesover developmental time, peaking just before flowering (Fig. 4).Genes that showed the reverse pattern were SPL8, SPL6, SPL3,SPL4, and SPL5 in A. thaliana, and SPL7 in A. lyrata, consistent witha role in the promotion of the juvenile to adult phase transition,which occurs after the production of four to six rosette leaves(Telfer et al., 1997). In contrast, A. thaliana SPL1, SPL12, SPL14,SPL2, SPL4, and SPL13 were expressed in leaves similarly acrossdevelopment, but transcript levels were at least 2-fold higher com-pared to the peak of A. lyrata leaf expression at both 19 (4 leaf stagevegetative plants) and 30 (12 leaf stage transitional plants) dayspost-germination. Data for A. thaliana were largely consistent withthe eFP Browser microarray data (http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi) where available (all but SPL6).

3.3. SBP-box gene expression in SAMs

With the exception of MgSBP1, which was similar between veg-etative and reproductive SAMs, expression of all sampled IM767SPL genes increased in SAMs following the transition to flowering(Fig. 3b). A similar pattern was observed for PR MgSPL5, MgSBP1,MgSPL7, and MgSPL8. Expression levels of MgSPL1, MgSPL2, MgSPL4and MgSBP2 were similar between vegetative and reproductiveSAMs of PR, with only MgSBP2 levels being at least 1.5-fold lowerin PR versus IM767 reproductive SAMs (Fig. 3b). However, expres-sion levels of MgSPL5, MgSBP1, MgSBP2, MgSPL7, and MgSPL8 wereat least 3-fold higher in vegetative SAM’s collected at leaf pair onein IM767 versus PR.

Fig. 2. Maximum likelihood (ML) phylogram showing SPL orthologs of Arabidopsis thaliana, A. lyrata, and Mimulus guttatus IM62, IM767, and PR. Arabidopsis and Mimulusgenes fall into eight previously described clades (Salinas et al., 2012; Preston and Hileman, 2013) and are listed to the right. Genes in bold were targeted for expressionanalyses. Clades that contain one or less SPL homolog from each Mimulus and Arabidopsis accession were collapsed. Black, gray, and open circles at nodes (or next to taxonnames for Mimulus and Arabidopsis genes in collapsed clades) represent ML bootstrap values >90%, >70%, and >50%, respectively. Since Genbank does not provide accessionnumbers for sequences shorter than 200 bp, newly generated SPL sequences for IM767 and PR can be found in Supplementary material (Fig. S1).

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Expression for the majority of SPL genes was positively corre-lated with the transition to flowering in both A. thaliana (19 d veg-etative SAM to 30 d inflorescence SAM) and A. lyrata (19 dvegetative SAM to 80 d inflorescence SAM), including SPL7, SPL1,SPL8, SPL6, SPL2, SPL3, SPL4, and SPL5 (Fig. 4). A similar increasein expression was observed for SPL14 in A. lyrata and SPL13 in A.thaliana SAMs; expression of the orthologs in A. thaliana and A.lyrata, respectively, were similar across stages. The only SPL genetranscript that became less abundant in SAMs following the transi-tion to flowering was SPL12 in A. thaliana (Fig. 4). For all genes, ex-cept AtSPL1 and AtSPL12, expression was at least 1.5-fold higher in30-day old inflorescence SAMs of A. thaliana compared to 30-dayold vegetative SAMs of A. lyrata. All A. thaliana data are fully consis-tent with the eFP Browser microarray data where available.

3.4. SBP-box expression in nodes bearing AMs

To test predictions of the hypothesis that differences in axillaryorgan identity/determinacy between IM767 and PR, and A. lyrataand A. thaliana are due to spatial differences in SPL gene expression,relative mRNA transcript levels were determined at basal nodesbearing as yet unexpanded AMs for all four taxa at the transitionto flowering. In IM767, MgSPL1, MgSPL2, MgSPL4, MgSBP1, andMgSPL7 mRNA transcripts were at a similar or higher level in nodeone AMs compared with inflorescence SAMs (Fig. 3b). Thus, sincenode one IM767 AMs are destined to produce primary axillaryinflorescences (n1 pai), and the expression of MgSPL1, MgSPL2,MgSPL4, MgSBP1, and MgSPL7 increases in inflorescence SAMs fol-lowing the transition to flowering, these data are consistent witha role for these genes in signaling reproductive phase change tomeristems (Fig. 5). In contrast, expression levels of IM767 MgSPL5,MgSBP2, and MgSPL8 were similar between node one axillary andvegetative SAMs, and at least 2-fold lower than in inflorescenceSAMs (Fig. 3b).

Unlike IM767, nodes one and two of PR either fail to expand (n1uam) or develop vegetative axillary branches (n2 vab), respectively(Fig. 1). In node one, the only transcripts that were similarly ormore abundantly expressed relative to inflorescence PR SAMs,were those whose expression did not positively correlate withthe floral transition in SAMs, namely MgSPL1, MgSPL2, and MgSPL4(Figs. 3b and 5). A similar trend was observed for MgSPL1, MgSPL2,MgSPL4, and MgSBP2 in node two. However, in the case of MgSPL2and MgSBP2, expression was at least 5-fold higher in node two thanin all other PR tissues, suggesting a possible role in axillary branchoutgrowth or repression of inflorescence identity (Fig. 3b).

Arabidopsis thaliana and A. lyrata, like IM767 and PR, differ inarchitecture. Whereas A. thaliana exerts strong apical dominance,resulting in lateral outgrowth only from a few AMs towards theapex, most A. lyrata AMs develop into axillary inflorescences(Fig. 1a and b). Consistent with the lack of axillary inflorescencedevelopment, all examined SPL genes were expressed at low levelsin basal unexpanded AMs (n4 uam) relative to inflorescence SAMsin A. thaliana. In contrast, with the exception of AtSPL8 and AtSPL3,which were both expressed at low levels, all SPL genes in A. lyratawere similarly or more strongly expressed in basal nodes withassociated AMs that would have eventually produced primary ax-illary inflorescences (n4 pai), relative to inflorescence SAMs (Fig. 4).Together with SAM expression data, these results support thehypothesis that spatial variation in SPL gene expression is impor-tant for architectural differences in Arabidopsis (Fig. 5).

4. Discussion

We cloned a representative subset of SPL genes in two pairs ofclosely related annual and perennial taxa, and found highly

variable expression patterns, suggesting functional diversificationfollowing both gene duplication and population/species divergence(Fig. 5). Expression for the majority of genes correlates with earlyand late flowering in SAMs of annual and perennial taxa, respec-tively. In many instances, differential expression in nodes coincideswith the identity and fate of associated AMs. However, a generallack of correlation between expression in leaves and floweringtime (Fig. 5) suggests that some SPL genes function early in devel-opment to promote phase change in Arabidopsis, and M. guttatusPR.

4.1. SPL gene functioning in Arabidopsis flowering time and phasechange

Previous studies have demonstrated that, of the functionallycharacterized A. thaliana SPL genes, at least six are involved inthe regulation of juvenile to adult phase change and the transitionto flowering (Stone et al., 2005; Wu and Poethig, 2006; Schwarzet al., 2008; Wang et al., 2009b; Yamaguchi et al., 2009). However,the fact that double and triple mutants of clade VIII and V genes,respectively, have more extreme phenotypes than single mutants(Schwarz et al., 2008; Shikata et al., 2009), suggests that this mightbe an underestimate. Consistent with the promotion of floweringhypothesis, gene expression in SAMs of A. thaliana is positively cor-related with flowering for 9 out of 11 tested genes. This includesthe previously uncharacterized genes AtSPL6 (clade IV) and AtSPL1(clade II), but precludes the negative regulator of flowering timeAtSPL14 (Stone et al., 2005) and its closely related paralog AtSPL12.Interestingly, with the exception of AlSPL13, expression of ortholo-gous SPL genes is positively correlated with flowering in SAMs of A.lyrata, supporting conservation of biochemical function across Ara-bidopsis (Fig. 5).

Despite the positive trend in SAMs, SPL gene expression in A.thaliana leaves is generally not correlated with flowering. Only AtS-PL7 shows a peak of expression in rosette leaves at bolting stage;no phase change or flowering time function has yet been assignedto this gene (Yamasaki et al., 2009). In the case of known or sus-pected promoters of flowering, including AtSPL3, AtSPL4, AtSPL5,AtSPL9, and AtSPL15, all genes also affect the juvenile to adult tran-sition (Wu and Poethig, 2006; Schwarz et al., 2008; Wang et al.,2000b; Yamaguchi et al., 2009). Thus, the lack of correlation be-tween leaf and SAM expression in A. thaliana is suggestive of strongpleiotropy in the regulation of early and late phase change. Alter-natively, early leaf expression could indicate a leaf developmentalfunction as has been described for genes in clades V, VII, and VIII(Schwarz et al., 2008; Shikata et al., 2009; Martin et al., 2010a,b).Unlike A. thaliana, leaf expression for about half of A. lyrata SPLgenes is positively correlated with the increasing trend of expres-sion in SAMs. This pattern is consistent with sub- and/or neo-func-tionalization of SPL genes in phase change and/or leaf developmentfollowing the split of Arabidopsis species less than three millionyears ago (Clauss and Koch, 2006). This hypothesis warrants futurefunctional testing.

4.2. Flowering time and SPL gene expression in Mimulus guttatus

Few studies have been conducted to determine the function ofSPL genes in asterid core eudicots. Exceptions are the clade VIgenes SlCNR and AmSBP1 in Solanum lycopersicum and Antirrhinummajus, respectively (Klein et al., 1996; Manning et al., 2006;Preston and Hileman, 2010), which are orthologous to AtSPL3,AtSPL4, and AtSPL5 in A. thaliana. Mutations in SlCNR result in fruitsthat fail to ripen (Manning et al., 2006). In contrast, silencing ofAmSBP1 results in non-flowering plants that lose apical dominance(Preston and Hileman, 2010). Although it is unknown whether slcnrmutants have altered phase change or flowering time, the timing of

Fig. 3. Expression of SPL homologs across a developmental series of M. guttatus leaves (a) and meristems (b) grown under long days. With the exception of the clade-VI genesMgSBP1 and MgSBP2 all genes numbers indicate SPL clade affiliations. Mean expression is based on 3–5 biological replicates with standard deviations. Black bars, IM767expression; gray boxed bars, PR expression. n1 pai, node one with an axillary meristem destined to become a primary axillary inflorescence; n1 uam, node one with anaxillary meristem that will remain unexpanded; n2 vab, node two with an axillary meristem destined to become a vegetative axillary branch; veg sam, apical vegetativemeristem; inf sam, apical inflorescence meristem.

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development in AmSBP1-silenced plants is only affected during thereproductive phase of growth. Thus, AmSBP1, AtSPL3, and probablyboth AtSPL4 and AtSPL5, promote flowering, but only the A. thalianagenes regulate vegetative phase change.

In annual M. guttatus IM767 the timing of SAM expression formost SPL genes positively correlates with flowering. The onlyexception is MgSBP1, which interestingly is the ortholog of theknown flowering time gene AmSBP1 (Klein et al., 1996; Preston

Fig. 4. Expression of SPL homologs across a developmental series of Arabidopsis leaves and meristems grown under long days. Mean expression is based on 3–5 biologicalreplicates with standard deviations. Black bars, A. thaliana expression; gray boxed bars, A. lyrata expression. 19 d lf, upper (youngest) leaf 19 days after germination; 30 d lf, upperleaf 30 days after germination; 80 d lf, upper leaf 80 days after germination; n4 uam, node four with an axillary meristem that will remain unexpanded; n4 pai, node four with anaxillary meristem destined to become a primary axillary inflorescence; 19 d veg sam, apical vegetative meristem 19 days after germination; 30 d veg sam, apical vegetativemeristem 30 days after germination; 30 d inf sam, apical vegetative meristem 30 days after germination; 80 d inf sam, apical inflorescence meristem 80 days after germination.

136 S.A. Jorgensen, J.C. Preston / Molecular Phylogenetics and Evolution 73 (2014) 129–139

Fig. 5. Spatial and temporal variation in SPL gene expression following geneduplication and population/species divergence. Relationships among clades arebased on Fig. 2. For leaves, open circles indicate that expression is not more than1.5-fold higher than other leaves, whereas filled circles indicate that expression is atleast 1.5-fold higher than other leaves. For shoot apical meristems (SAMs), openboxes indicate that expression is not more than 1.5-fold higher than other SAMs,whereas filled boxes indicate that expression is at least 1.5-fold higher than otherSAMs. For nodes and associated axillary meristems (AMs), open stars indicate thatexpression is not significantly higher than vegetative (veg) or inflorescence (inf)SAMs, whereas filled stars indicate that expression is significantly higher thanvegetative or inflorescence SAMs.

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and Hileman, 2010). In contrast, only half of sampled SPL genes inthe perennial PR population have SAM expression patterns thatpositively correlate with flowering. This might suggest that severalSPL genes in PR have no function in, or delay, flowering time. Alter-natively, if the PR population is derived from an early flowering an-nual population, early expression of several SPL genes in SAMsmight indicate retention of the ancestral expression pattern. Ongo-ing systematic studies to resolve relationships within the M. gutt-atus complex will help to distinguish between these hypothesesby determining the direction of transitions in growth habit.

In leaves, all but two SPL genes (MgSPL1 and MgSPL2) are ex-pressed in a manner consistent with the promotion of floweringtime in IM767. Thus, unlike A. thaliana, expression of most IM767SPL genes in leaves does not support a secondary function in juve-nile to adult phase change. This is consistent with functional stud-ies on AmSBP1 (Preston and Hileman, 2010). Although the peak ofMgSPL1 (clade I) and MgSPL2 (clade II) expression in IM767 coin-cides with flowering in SAMs, expression in leaves is upregulatedin vegetative plants prior to the floral transition. Molecular analy-ses in A. thaliana, tomato, and rice (Oryza sativa) have demon-strated that clade-I and -II SPL genes, whose coding regions arecharacteristically longer than other SPL clade genes, are not nega-tively regulated by miR156 in leaves (Gandikota et al., 2007;Rhoades et al., 2002; Salinas et al., 2012; Schwab et al., 2005;

Wu and Poethig, 2006; Xie et al., 2006). This might explain whythe expression of these genes peaks prior to flowering in leavesbut not SAMs.

In PR expression of MgSPL1 and MgSPL2 in leaves is also nega-tively correlated with flowering, reinforcing the hypothesis thatclade-I and -II genes lack negative regulation by miR156, and sug-gesting a possible role in promoting vegetative phase change. Asimilar negative trend is observed for MgSPL8 leaf expression.MgSPL8 is orthologous to AtSPL9 and AtSPL15, both of which pro-mote vegetative phase change and flowering, and delay leaf initia-tion rate in A. thaliana. Microarray (http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi) and q-RT-PCR (Schwarz et al., 2008) data have re-vealed that AtSPL9 and AtSPL15 expression peaks in SAMs duringand following the floral transition, and whereas AtSPL9 is upregu-lated in leaves, AtSPL15 retains a moderate level of leaf expressionthroughout development. Thus, the very early spike in MgSPL8expression in PR leaves represents a novel pattern of expression.It will be interesting to determine what the functional significanceof this is for PR development.

4.3. SPL gene expression correlates with evolution of growth habit andarchitecture

Plants have evolved the ability to synchronize flowering timewith locally favorable environmental conditions through changesin the spatio-temporal regulation of conserved flowering timegenes (Kemi et al., 2013), switches in flowering gene function(Pin et al., 2010), and/or deployment of novel developmental geneswithin the flowering time pathway (Yan et al., 2004). However, formost plant clades the genetic basis for both intra- and inter-spe-cific differences in flowering time is not well understood. In thelight of previous functional studies in A. thaliana and A. majus,our results reveal candidate SPL genes whose temporal expressionis consistent with flowering time differences between annual andperennial forms of Arabidopsis and M. guttatus.

Despite being upregulated in SAMs during the floral transitionof both Arabidopsis species, gene expression of most SPL genes ishigher in A. thaliana versus A. lyrata 30-day old SAMs, and peaks la-ter in A. lyrata. Thus, differential temporal regulation of SPL genes,either through changes in SPL regulatory elements or upstreamregulators, might have been important for flowering time differ-ences across Arabidopsis. In M. guttatus IM767 and PR this patternis also observed for MgSPL5, MgSPL7, and MgSPL8. However,MgSPL1, MgSPL2, MgSPL4, and MgSBP2 only correlate with flower-ing in IM767. If the ancestral M. guttatus population was an earlyflowering annual, the relatively early expression of MgSPL1,MgSPL2, MgSPL4, and MgSBP2 in PR might reflect a conservedexpression pattern in SAMs. In contrast, we hypothesize that de-layed expression of MgSPL5, MgSPL7, and MgSPL8 in PR has beenimportant for shifts from annuality to perenniality. This hypothesiscould be functionally tested by expressing SPL alleles from IM767in transgenic SPL-silenced lines of PR and vice versa, and by deter-mining the direction of growth habit transitions in the M. guttatusspecies complex.

In addition to temporal variation between taxa, spatial differ-ences in SPL gene expression correlate with different patterns ofreproductive branching architecture in Arabidopsis and to a lesserextent M. guttatus. In M. guttatus, which varies in AM outgrowthand identity, transcript levels of MgSBP1 and MgSPL7 are similarbetween AMs destined to become axillary inflorescences andboth vegetative and inflorescence SAMs of IM767, but are severalfold higher in inflorescence SAMs versus AMs of PR. The tempo-ral expression of both of these genes in IM767 and PR is consis-tent with them having a role in inflorescence development, ashas been functionally demonstrated for the A. majus MgSBP1ortholog AmSBP1 (Preston and Hileman, 2010). Additionally,

138 S.A. Jorgensen, J.C. Preston / Molecular Phylogenetics and Evolution 73 (2014) 129–139

MgSPL2 transcripts are much more abundant in stolon producingAMs (node two of PR) compared to all other Mimulus meristems.The MgSPL2 ortholog AtSPL14 in Arabidopsis is a negative regula-tor of inflorescence identity (Stone et al., 2005). Thus, our work-ing hypotheses are that MgSPL2 acts to repress inflorescenceidentity once branches have been initiated by genes such asMORE AXILLARY GROWTH (MAX) (Baker et al., 2012), and thatchanges in the spatial regulation of MgSPL2 underlie differencesin AM identity between IM767 and PR. This hypothesis predictsthat MgSPL2 constitutive expression in IM767 and silencing in PRwould result in loss and gain of axillary inflorescences,respectively.

In Arabidopsis, all rosette leaf-associated AMs have the potentialto form axillary inflorescences following release of dominance bythe apical inflorescence (Stirnberg et al., 2002). However, the pro-portion of lateral inflorescences that develop varies considerablybetween ecotypes and species, averaging around 39% in A. thalianaCol-0 and almost 100% in A. lyrata (Stirnberg et al., 2002). Of theknown or putative promoters of Arabidopsis flowering, SPL7, SPL1,SPL2, SPL4, and SPL5 are good candidates for inflorescence identityspecification in AMs based on high levels of expression in A. lyrata,but not A. thaliana, AMs.

Differences in A. thaliana branch outgrowth have been associ-ated with variation at the MAX2, MAX3, SUPERSHOOT1 (SPS1), andAGAMOUS-LIKE 6 loci (Tantikanjana et al., 2001; Ehrenreich et al.,2007; Huang et al., 2012), and mutant analyses have implicatedseveral other genes in AM formation and inhibition of branch out-growth, including BRANCHED1 (BRC1) (Aguilar-Martínez et al.,2007). Recently it was demonstrated that A. thaliana axillarybranch identity might occur after the initiation of shoot outgrowththrough BRC1 interference of FT and TWIN SISTER OF FT (TSF)(Niwa et al., 2013). This is consistent with previous reports ofAMs being initially insensitive to flowering signals (Hempel andFeldman, 1994; Grbic and Bleecker, 1996). However, since SPLgenes are generally thought to function upstream of FT, our datatentatively suggest that branch suppression can also occur inde-pendently of BRC1.

4.4. Perspective and future directions

Data from this and other studies predict an important role forSPL genes in juvenile to reproductive phase change in angiosperms.We found that heterochronic and heterotopic changes in MimulusMgSPL5, MgSBP1, MgSBP2, MgSPL7, and MgSPL8, and ArabidopsisSPL7, SPL6, SPL2, SPL4, and SPL5 expression patterns are consistentwith flowering time variation, and hypothesize a role for MgSPL2 inrepression of inflorescence identity in expanding axillary meris-tems. Given the contribution of flowering time variation and axil-lary branch identity to local adaptation, it will be particularlyinteresting to determine if any of these candidate genes fall withinquantitative trait loci for life history traits. Future functional stud-ies will also be required to assess the different developmental rolesof these genes in Mimulus and Arabidopsis taxa with variablegrowth habits.

Acknowledgment

This work was funded by the College of Agriculture and Life Sci-ence at the University of Vermont.

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.ympev.2014.01.029.

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