ORIGINALRESEARCH published:30October2015 doi:10.3389/fpls.2015.00931 Conservation of the abscission signaling peptide IDA during Angiosperm evolution: withstanding genome duplications and gain and loss of the receptors HAE/HSL2 IdaM.Stø1†,RussellJ.S.Orr1†,KimFooyontphanich2,XuJin3,JonfinnM.B.Knutsen1, UrsFischer3,TimothyJ.Tranbarger2,IngerNordal1andReidunnB.Aalen1* 1SectionforGeneticsandEvolutionaryBiology,DepartmentofBiosciences,UniversityofOslo,Oslo,Norway,2UMR Editedby: DiversitéetAdaptationetDéveloppementdesPlantes,InstitutdeRecherchepourleDéveloppement,Montpellier,France, MariaCarlotaVazPatto, 3DepartmentofForestGeneticsandPlantPhysiology,UmeåPlantScienceCentre,SwedishUniversityofAgricultural UniversidadeNovadeLisboa, Sciences,Umeå,Sweden Portugal Reviewedby: ThepeptideINFLORESCENCEDEFICIENTINABSCISSION(IDA),whichsignalsthrough ChadNiederhuth, UniversityofGeorgia,USA the leucine-rich repeat receptor-like kinases HAESA (HAE) and HAESA-LIKE2 (HSL2), CoralieLashbrook, controls different cell separation events in Arabidopsis thaliana. We hypothesize the DesMoinesAreaCommunityCollege, involvementofthissignalingmoduleinabscissionprocessesinotherplantspecieseven USA JohnWalker, though they may shed other organs than A. thaliana. As the first step toward testing UniversityofMissouri,USA this hypothesis from an evolutionarily perspective we have identified genes encoding *Correspondence: putative orthologs of IDA and its receptors by BLAST searches of publically available ReidunnB.Aalen [email protected] protein, nucleotide and genome databases for angiosperms. Genes encoding IDA or †Jointfirstauthors. IDA-LIKE(IDL)peptidesandHSLproteinswerefoundinallinvestigatedspecies,which wereselectedastorepresenteachangiospermorderwithavailablegenomicsequences. Specialtysection: The12aminoacidsrepresentingthebioactivepeptideinA.thalianahavevirtuallybeen Thisarticlewassubmittedto CropScienceandHorticulture, unchanged throughout the evolution of the angiosperms; however, the number of IDL asectionofthejournal andHSLgenesvariesbetweendifferentordersandspecies.Thephylogeneticanalyses FrontiersinPlantScience suggest that IDA, HSL2, and the related HSL1 gene, were present in the species that Received:14July2015 gave rise to the angiosperms. HAE has arisen from HSL1 after a genome duplication Accepted:15October2015 Published:30October2015 that took place after the monocot—eudicots split. HSL1 has also independently been Citation: duplicated in the monocots, while HSL2 has been lost in gingers (Zingiberales) and StøIM,OrrRJS,FooyontphanichK, grasses(Poales).IDAhasbeenduplicatedineudicotstogiverisetofunctionallydivergent JinX,KnutsenJMB,FischerU, IDL peptides. We postulate that the high number of IDL homologs present in the core TranbargerTJ,NordalIandAalenRB (2015)Conservationoftheabscission eudicots is a result of multiple whole genome duplications (WGD). We substantiate the signalingpeptideIDAduring involvementofIDAandHAE/HSL2homologsinabscissionbyprovidinggeneexpression Angiospermevolution:withstanding genomeduplicationsandgainand dataofdifferentorganseparationeventsfromvariousspecies. lossofthereceptorsHAE/HSL2. Front.PlantSci.6:931. Keywords:LRR-RLK,phylogeny,genomeduplication,peptidesignaling,leafabscission,fruitabscission,Populus, doi:10.3389/fpls.2015.00931 oilpalm FrontiersinPlantScience|www.frontiersin.org 1 October2015|Volume6|Article931 Støetal. EvolutionaryconservationoftheIDApeptide INTRODUCTION only IDA and AtIDL1 can fully complement the ida mutant phenotype. Together this may suggest that AtIDL2-5 peptides Plant architecture is dependent on the balance between cell haveloweraffinitytoHAEandHSL2,andpossiblysignalthrough division and cell elongation, proliferation and differentiation, (a)different,butrelatedreceptor(s)(Stenviketal.,2008).There as well as formation and abscission of organs. Plants are are more than 600 genes in A. thaliana encoding receptor-like sessile organisms that over evolutionary time have adapted to kinases,andofthesemorethan200encodeextracellulardomains theirenvironment.Theyhavedevelopedreproductivestrategies with LRRs of varying length assumed to bind small peptides involving mechanisms that in various ways facilitate fruit (ShiuandBleecker,2001).Onlyaboutadozenofthesehavebeen and seed dispersal. The shedding of organs that have served matched with a ligand (Butenko et al., 2009). The majority of their purpose is furthermore part of normal development, but these,includingHAEandHSL2,belongtotheRLKsubclassXI, detachment can also be a response to injury or environmental whichhasmorethan20LRRs. changes. The model plant A. thaliana shed individual floral Interestingly, ectopic expression of IDA in A. thaliana leads organs (petals, sepals, and stamen) shortly after pollination to abscission of fruit, flowers, branches, and cauline leaves at (BleeckerandPatterson,1997),anddisplaysopeningofthevalves vestigial AZ at the base of these organs, which are sites of cell ofthematuresiliques(socalleddehiscence),andthereafterseed separationinanumberofotherspecies,butnormallynotinA. dropping. Other species may shed leaves (e.g., deciduous trees thaliana(Stenviketal.,2006).Additionally,theIDA-HAE/HSL2 likeaspen,Populustremula)andwholeflowers(e.g.,Citrus),or signaling module is involved in a quite different cell separation fruits(e.g.,tomato,palms). process, namely lateral root emergence (Kumpf et al., 2013). Despitevariationinthesitesofabscissionindifferentspecies IDAandthereceptorsareexpressedintheendodermal,cortical, of flowering plants, various abscission events are similar at the and epidermal cells overlaying lateral root primordia, induce cellularlevelinthattheytakeplaceinspecializedabscissionzones expressionofcellwallremodelinggenesandfacilitateemergence (AZs).AZsareeithergeneratedalreadyduringthedevelopment without cellular disruption (Aalen et al., 2013; Kumpf et al., of the organ, or can be induced in response to hormonal 2013). or environmental cues (Patterson, 2001; Roberts et al., 2002; The objective of the presented work has been to investigate Lewis et al., 2006; Aalen, 2011). Organ detachment is a cell towhatextentorthologsofIDA,HAE,andHSL2arepresentin separationprocessthatinvolvesdissolutionofthemiddlelamella other angiosperms irrespective of which organs they shed, and betweenadjacentAZcellfilespresumablythroughtheactionofa whether gene expression data could substantiate a role of these numberofdifferentcellwallremodelinganddegradingproteins, orthologsincellseparationevents. e.g., polygalacturonases, expansins, XTHs, and endoglucanases (Cho and Cosgrove, 2000; González-Carranza et al., 2007; Lashbrook and Cai, 2008; Meir et al., 2010; Liu et al., 2013; MATERIALS AND METHODS Niederhuth et al., 2013; Tsuchiya et al., 2015). One can assume that there is a need for tight regulation and coordination of Phylogenetic Analyses expression of genes involved in cell separation processes, since Sequences used in this study were obtained from various cellwallweakeningandbreakdowncouldrendertheplantmore databases [NCBI-protein, NCBI-Assembly, Phytozome, susceptibletopathogenattack,andprematureorganlosswould Ancestral Angiosperm Genome Project (AAGP), Comparative reducereproductivesuccess. Genomics (CoGe)] using either tBLASTn or BLASTp (as of InA.thalianaapeptideligand—receptorsystemresponsible 3.2015) with an A. thaliana IDL or HSL query. Outgroup for such tight regulation of floral organ abscission has been taxa for both monocot and eudicot receptors were closely identified (Butenko et al., 2003; Stenvik et al., 2008). This related A. thaliana representations within the subclass XI signaling module consists of the secreted peptide IDA encoded LRR-RLKs. IDA was inferred without an outgroup. An aa by the gene INFLORESCENCE DEFICIENT IN ABSCISSION alignment(∼59aa)ofangiospermIDAwithouttheN-terminal (At1g68765), and the two leucine-rich repeat (LRR) receptor- secretion signal and LRR-RLK group (full length 1926 aa) was like kinases (RLK) HAESA (HAE) and HAESA-LIKE2 (HSL2) constructed using MAFFTv6 E- INS-I model under default (Choetal.,2008;Stenviketal.,2008).Inbothidaandhaehsl2 settings (Katoh and Toh, 2008). The resulting alignments were mutants floral organs are retained indefinitely. IDA belongs to checked with Gblocks v0.91b (Castresana, 2000), under the a small gene family in A. thaliana with five additional IDA- least stringent parameters (small final block, gap positions in LIKE (AtIDL) members that to a varying degree can substitute final block and less strict flanking), to exclude poorly aligned for IDA function (Butenko et al., 2003; Stenvik et al., 2008). positions and divergent regions from subsequent phylogenetic IDA and the IDL proteins share a short C-terminal sequence inferences.MultiplehomologouscopiesofHSL1/HSL2/HAESA encompassingthebioactivepeptide(Stenviketal.,2008),which (At1g28440/At5g65710/At4g28490) for each species were forIDAhasbeendelineatedtoa12aminoacids(aa)longproline- removed to reduce phylogenetic noise; in all cases the copy richpeptide(hereaftercalledmatureIDA,mIDA)usinggenetic with most sequence data and thereafter shortest branch length andbiochemicaltools(Stenviketal.,2008;Butenkoetal.,2014). was retained for further analysis. ProtTest v2.4 (Abascal et al., Overexpression either of IDA or AtIDL genes, results in early 2005) determined LG as the optimal evolutionary model. abscissionandenlargedabscissionzones(AZ)atthebaseofthe Maximum Likelihood (ML) analyses were performed with floral organs, but when expressed using IDA’s own promoter, RAxML-VI-HPCv8.0.26,usingthePROTCATLGmodelwith25 FrontiersinPlantScience|www.frontiersin.org 2 October2015|Volume6|Article931 Støetal. EvolutionaryconservationoftheIDApeptide ratecategories(Stamatakis,2006).Themostlikelytopologywas angiosperms,monocots,basaleudicots,andcoreeudicotclades established from 100 separate searches and bootstrap analyses wereusedtodeterminethephylogeneticrelationship. wereperformedwith500pseudoreplicates. The LRR-RLK ingroup is monophyletic, separated from the All model estimation and phylogenetic analyses were done outgroup with high support (98% Bootstrap, BP) (Figure1, using either Lifeportal (https://lifeportal.uio.no) or the abel SupplementaryFigureS1).HSL2andHSL1/HAErepresenttwo server at the University of Oslo. All alignments constructed fullysupporteddistinctclades(100%BP).IntheHSL2clade,the as part of this study are available as Supplementary Data sets, basalangiospermcopyisfirsttodivergewithinafullysupported and will in addition be made freely available as Supplementary grouping,leavingahighlysupported(92%BP)monocot/eudicot resources through the authors’ Research Gate pages (https:// clade.Subsequently,amoderatelysupported(71%BP)monocot www.researchgate.net/profile/Reidunn_Aalen) and (https:// clade diverges, leaving a poorly supported (54% BP) eudicot www.researchgate.net/profile/Russell_Orr2). monophyly.HSL2ishowevermissinginthetaxafromtheorders PoalesandZingiberales(SupplementaryFigureS1). Plant Material The HSL1/HAE clade is fully supported with the basal A. thaliana has been transformed with HAE and HSL2 angiosperm copy being first to diverge leaving a moderately promoter::GUS constructs in the vector pMDC162 made using supported (84% BP) monocot/eudicot clade (Figure1). The Gateway Cloning Technology (Curtis and Grossniklaus, 2003) monocot HSL1 copy diverges next forming a highly supported withthesamepromoterregionupstreamofthecodingsequence clade (96% BP). Two groupings are found within the monocot (1601 bp and 2300 bp, respectively) as previously reported for clade; the first—constituting all monocot orders (HSL1A)—is HAE and HSL2 constructs with YFP reporter (Kumpf et al., unsupported (40% BP), but excluded from the second, longer- 2013). branching HSL1B. This duplicate HSL1 clade for the orders Fully expanded leaf blades of hybrid aspen, Populus tremula Arecales(palms),Poales,andZingiberales,hasmoderatesupport L.XP.tremuloidesMichx.;cloneT89,wereshadedinaluminum (72% BP) (Figure1, Supplementary Figure S1). The eudicot foiltoinduceabscissionandtotalRNAwasextractedfrom3mm- HSL1/HAE grouping is moderately supported (70% BP). The thick leaf axils 6 days after shading started using RNeasy Plant HAE clade, representing Vitales in addition to all Rosids and MiniKit(Qiagen).TwomicrogramsoftotalRNAwasusedasa Asterids, is moderately supported (80% BP) and excludes the template for reverse transcription with the QuantiTect Reverse basaleudicotordersRanunculales,Proteales,andCaryophyllales TranscriptionKit(Qiagen).Quantitativereal-timePCR(qPCR) which have a closer affinity to the moderately supported (82% was performed using SYBR Green I Master in combination BP)eudicotHSL1grouping.ThefullysupportedCaryophyllales withaLC4800(RocheDiagnostics)qPCRmachine.Primersare cladeformsamoderatelysupported(80%BP)monophylywith specifiedinSupplementaryTableS1.Expressionwasnormalized theeudicotHSL1grouping. toPtACTIN1expression. OuranalysesrevealedthatallangiospermshaveHSL1genes, mRNA was isolated from AZs of unripe and ripe of oil in in basal angiosperms and some monocots, and almost all palm (Elaeis guineensis) fruit, and AZs of fruit treated with eudicots in combination with a HSL2 gene. Only the core ethylene as described previously (Roongsattham et al., 2012). eudicots have both a HAE and a HSL2 gene. In A. thaliana Primers were designed to fit the various EgIDA genes, EgHSL2, HAEandHSL2areredundantinfunctionwithrespecttofloral andEgHSL1(SupplementaryTableS1).qPCRwasperformedas organ abscission, as both genes must be mutated to give the previouslydescribed(Roongsatthametal.,2012).Allexpression abscission-deficient phenotype (Cho et al., 2008; Stenvik et al., was normalized to the EgEfα1 (accession number: AY550990) 2008). expression. No change of EgEfα1 transcript accumulation was HAE and HSL1 Differ from HSL2 Especially found in the fruit tissues treated or not treated with ethylene. in their Kinase Domains ControlswereconductedtovalidatetheabsenceofDNAineach sample. LRR-RLK proteins consist of three domains with different functions: the intracellular kinase domain that confers the signaling output upon ligand binding, the transmembrane RESULTS domainthatanchorsthereceptorsintheplasmamembrane,and the extracellular LRR to which the ligand will bind (called the HSL2 has been Lost in the Gingers and the ectodomain). As these three domains have different functions, Grasses, and HAE is Present only in theselectivepressureforrejectingorpreservingnewmutations Eudicots mightdiffer. The A. thaliana HAE and HSL2 receptors are closely related The intracellular kinase domain has typically two regions: (60% similar and 45 % identical aa) but HAE is even more the N-terminal lobe with several β strands and a conserved closely related to HSL1 (73% similarity, 58% identical aa), an ATPbindingsite,andthelargerC-terminalpartwithanumber RLK so far with unknown function. BLASTp and tBLASTn of α-helices, and an activation loop with serine/threonine were used to identify putative HSL1 angiosperm orthologs in [Ser(S)/Thr(T)] target residues for phosphorylation. In the additiontoorthologsofHAEandHSL2.Otherrelatedreceptors BRASSINOSTEROID INSENSITIVE1 (BRI1) LRR-RLK from the LRR-RLK subclass XI were used as outliers. Protein several such residues are phosphorylated (Wang et al., sequencesfrom60speciesbelongingto24orderscoveringbasal 2005). These residues are well conserved in the A. thaliana FrontiersinPlantScience|www.frontiersin.org 3 October2015|Volume6|Article931 Støetal. EvolutionaryconservationoftheIDApeptide FIGURE1|PhylogenyofHSLLRR-RLKevolutionwithinAngiosperms.PhylogenyinferredfromMLwith145ingrouptaxaand784aminoacidcharacters.The phylogenyhasbeencollapsedatdifferenttaxonomiclevelsandshowsonlybootstrapvalues>50.TheexpandedtreeispresentedinSupplementaryFigureS1,which includesanoverviewofspeciescomprisingeachorder.ThealignmentsusedfortheconstructionofthisphylogenyareavailableasSupplementaryDataSheet1and2. HAE, HSL1 and HSL2 receptors and their orthologs in residues are interesting since selection pressure conserving other species (Supplementary Figure S2A). This includes the residues involved in ligand binding is likely. To identify such residue corresponding to S861 of HAE, which is subject to residuesweemployedtheRepeatConservationMapping(RCM) autophosphorylation (Taylor et al., 2013), and several residues (http://144.92.198.58/main/main.php), which relies on a set of predicted to be targets of Ser/Thr kinases (e.g., by using algorithmstoidentifypredictedfunctionalsitesofLRRdomains. PhosPhAt http://phosphat.uni-hohenheim.de/) (Durek et al., This is accomplished by identifying the extent of conservation 2010). However, in loops between helices positions of putative of different aa patches on the predicted surface of LRR and phosphorylation sites differ in HSL2 receptors compared to generatingacoloredheatmap,displayinghowconservedanaa HAE/HSL1. Furthermore, HSL2 receptors lack a C-terminal is in a given position in a set of orthologs (Helft et al., 2011). extension with putative phosphorylation sites (Supplementary ForalltheHSLreceptorsaregionwithhighlyconservedaawas Figure S2A). The predicted secondary structures of the stretching from the seventh LRR with the common signature transmembrane domains of HAE, HSL1, and HSL2 are similar, sequence QIEL[Y,F], to the fourteenth LRR (Figure2). The with a membrane-spanning, hydrophobic α-helix flanked by signaturesweremoresimilarbetweentheHAEandHSL1,than conserved aa (Supplementary Figure S2B). HSL2 sequences HSL2. differfromHAEandHSL1sequencesbyashorterα-helix.HAE The Bioactive IDA Peptide is Conserved in sequencescanbedistinguishedfromHSL1byatwoaadeletion near the end of the transmembrane domain (Supplementary all Flowering Plants FigureS2B). IDA and IDL are secreted peptides generated from So far, the exact location of the A. thaliana mIDA peptide prepropeptides (Figure3A), where the hydrophobic N- when binding HAE or HSL2 is not known, however, in other terminus is a signal directing the protein to the secretory casessmallpeptideshavebeenshowntobindconsecutiverepeats pathway. The variable middle part and the C-terminus are ontheinsidepocketoftheLRR(Sunetal.,2013).Eachrepeathas assumed to be cleaved off in the apoplastic space to release a typically24residueswithsixevenlydistributedconservedleucine mature 12 aa peptide named PIP after the three first residues, residues(Leu,L),whichtogetherwithasparagine(Asn,N)and PIPPSAPSKRHN(Butenkoetal.,2003).SyntheticmIDApeptide glycine (Gly, G) in conserved positions build the structural withhydroxylationonthecentralproline(Pro,P)canbindand framework of the ectodomain (Figure2). These residues are activate HSL2 efficiently, and HAE at a higher concentration almostinvariableandhavethereforelimitedinformativevaluein (Butenko et al., 2014). The Pro residues in positions 2, 3, aphylogeneticperspective.Incontrast,conservednon-structural and 7, serine (Ser, S) in positions 5 and 10, and histidine FrontiersinPlantScience|www.frontiersin.org 4 October2015|Volume6|Article931 Støetal. EvolutionaryconservationoftheIDApeptide FIGURE2|Continued andsimilarityofaaresiduesinagivenposition(Xaxis)inagivenrepeat(Y-axis) onthesurfaceoftheLRRdomainofHAEorthologsfromtheeudicots,HSL1 fromeudicots,HSL1AandBfrommonocots,HSL2fromeudicotandfrom monocots.Theheatmapsaregeneratedusingthesameorthologsequences asusedforthephylogeneticanalysis(SupplementaryFigureS1).The alignmentsusedforheatmapconstructionareavailableasSupplementary DataSheet2. (His, H) asparagine (Asn, N) at the end are found in all A. thaliana IDA/IDL peptide sequences. Experiments where the part of the AtIDA gene encoding PIP was swapped with the correspondingAtIDLsequenceandtherecombinantAtIDA-IDL gene introduced in the ida mutant, indicate the importance of different aa residues (Stenvik et al., 2008). The AtIDL1 peptide (LVPPSGPSMRHN) complements the mutation, suggesting thattheinitialProisoflittleimportance,thatthehydrophobic isoleucine (Ile, I) and the small central Ala residues can be exchanged with the hydrophobic valine (Val, V) and the small Gly, respectively, without affecting the biological activity, and furthermore that a positively charged aa in position 9 is no absoluterequirement.Incontrast,AtIDL2,AtIDL3,AtIDL4,and AtIDL5cannotfullycomplementtheidamutation(Stenviketal., 2008). Their PIP motifs are characterized by arginine-lysine (ArgLys,RK)inposition9and10,incontrasttotheLysArg(KR) foundinmIDA. BLAST and tBLASTn searches were executed using the C- terminalsequencesofA.thalianaIDAandIDL(i.e.,PIPextended with eight aa N-terminally, and the C-terminal aa, Figure3A) BLASThitswerecuratedforlengthofthecodingregion(<120 aa), presence of hydrophobic secretion signal, the C-terminal position of the conserved PIP motif, and the presence of the conserved Pro, Ser, and HisAsn residues. IDA and/or IDL sequenceswereidentifiedinallgroupsinourdatasets. We were unable to infer a phylogeny over the evolution of IDA, due to the short sequence of the conserved peptide. IDA lacks phylogenetic information and is also present in a high number of copies in many species; any possible pattern was unclear, with no clear clades, branching patterns, and a general lack of stability. Despite this we attempted an inference based on multiple datasets (PIP, EPIP, and EPIP- C, Figure3A), considering species that have undergone fewer genome duplications (Vanneste et al., 2014), however, without success. As an alternative approach to understand the evolution of IDA/IDL peptides, we grouped the different PIP motifs of the BLAST-identified proteins based on their amino acid composition. In all eudicots, peptides could be classified either as IDA/IDL1 ([P,L][V,I]PPS[A,G]PSKRHN) or like IDL2-5 (PIP[A,T,H,P]S[A,G]PSRKHN), named PIP and PIP R K respectivelybasedontheresidueinposition10(Figures3B,C). Both motifs were found in every eudicot species tested. In FIGURE2|ConservedaminoacidsintheLRRofHSLproteins. the monocot dataset PIP was found in the orders Arecales R HeatmapsgeneratedusingRepeatConservationMapping(RCM)ofLRR and Zingiberales. Additionally, we identified a version in all domains(http://144.92.198.58/main/main.php)reflectingdegreeofidentity monocots with close resemblance to PIP , but containing a (Continued) R glutamine(Gln,Q)inposition10(PIP ,Figure3D). Q FrontiersinPlantScience|www.frontiersin.org 5 October2015|Volume6|Article931 Støetal. EvolutionaryconservationoftheIDApeptide benthamiana and tomato that are more similar within than betweenthespecies(SupplementaryFigureS3A),suggestingthat additional independent duplications have taken place after the divergenceofNicotianoideaeandSolanoideae.Similarly,wehave previously suggested that the AtIDL2 and AtIDL3 genes in A. thaliana result from a recent duplication event (Stenvik et al., 2008). IDA Ligands and HSL Receptors are Expressed in a Variety of AZs and Species HavingidentifiedorthologsofIDAanditsreceptorsinavariety of species, we examined in the literature and experimentally whether IDA/IDL1 and the relevant receptors were likely to be involvedincellseparationevents. FloralOrganandFlowerAbscission Abscission plays an important role during plant reproduction. It is common among eudicots (including A. thaliana) to abscise turgid floral organs after pollination, when they no longer are functional (van Doorn, 2001). In some species the function of organs change during development, exemplified by tepals in Eriophyllum spp. (Asterales) and Polygonum spp. (Caryophyllales) that first act as protection of reproductive organs and later as organs involved in seed dispersal. In monocots floral organs may wither without abscission (van Doorn, 2001, 2002), or the perianth is retained and will cover the fruits during seed maturation. Hence, in such settings IDA and its receptors are not expected to be active. We postulate thatthesignalingsystemisintactinmonocotsinspecieswhere FIGURE3|IDAandIDLpeptides.(A)StructureofIDAandIDL abscission of individual organs takes place when the tepals prepropeptides.(B–D)Peptideconsensussequencesasindicated.The alignmentusedfortheconstructionofthepeptidelogosisavailableas are free, like in tulips (Tulipa spp., Liliales). Unfortunately SupplementaryDataSheet3. genomic and transcriptomic data are scarce for many monocots, especially the Liliales which have gigantic genomes (Shahinetal.,2012). The IDL Gene Family has Expanded in Normally A. thaliana does not shed cauline leaves, whole Many Taxa of Core Eudicots flowers or fruits. However, ectopic expression of IDA and About 65 million years (Myr) ago after the split of the core IDL peptides in A. thaliana leads to induction of abscission eudicots into Rosids and Asterids, whole genome duplication in a HAE/HSL2-dependent manner at the base of pedicels, (WGD)tookplaceinanumberofeudicotlineagesandexpanded caulineleaves,andinflorescencebranches,ratherthanageneral both the number of species and the gene number of each separation of cells in the plant (Figures4A–D) (Stenvik et al., species (Van de Peer et al., 2009). In line with this, detailed 2006).Thesearesitesofabscissioninotherspecies.Interestingly, inspection suggests independent evolution especially of the A.thalianalinestransformedwithpromoter:GUSconstructsfor IDL2-5 genes in these two clades. Soybean (Glycine max), HAEorHSL2showthatthereceptorgenesareexpressedinthe common bean (Phaseolus vulgaris) and A. thaliana belong to vestigialAZsatthesesites(Figures4E–G). the Rosids, the beans representing the families of legumes Fall of whole flowers is less common than floral organ (Fabales),andA.thalianabelongingtotheBrassicales.Soybean abscission(Figure5A),butoccursinbothmonocotandeudicot has six pairs of IDL genes, each pair represented only once species,whenpollinationorfertilizationfail(vanDoorn,2002). in common bean, consistent with a late genome duplication Abscission of immature fruit is a normal event in several in soybean after the separation from bean (Tucker and cultivated species. In Citrus spp. (Sapindales), abscission of Yang, 2012). Tomato (Solanum lycopersicum), potato (Solanum flowers and young fruit results from cell separation at an AZ tuberosum), and tobacco (Nicotiana ssp.) belong to the family at the base of the floral pedicel. The CicIDA3 gene, expressed Solanales in the Asterid clade, and are considered diploid in Citrus clementina AZs, encodes a protein with a PIP motif species. However, analyses of inter- and intrachromosomal identical to mIDA with the exception of a Gly instead of an duplicationsinthetomatoandpotatogenomessuggestasecond Alainposition6(Estornelletal.,submitted).Overexpressionof more recent WGD in the common ancestor of Petunoideae, CicIDA3 in A. thaliana induced the same phenotypic changes Nicotianoideae, and Solanoideae (Song et al., 2012). We as have been shown for overexpression of IDA and AtIDL1 have found pairs of almost identical IDL genes both in N. (Figures4A–D)(Estornelletal.,submitted),suggestingthatthe FrontiersinPlantScience|www.frontiersin.org 6 October2015|Volume6|Article931 Støetal. EvolutionaryconservationoftheIDApeptide FIGURE4|ExpressionofHAEandHSL2promoter:GUSconstructsat sitesofectopicabscission.(A–C)EnlargementofAZandpremature FIGURE5|Modesofabscission.(A)Abscissionof(a)sepals,(b)petals,(c) abscissionofwholeflowersandimmaturefruitscomparedtowildtypesilique stamen,and(d)carpels.(B)Abscissionofleavesattheaxilofthepedicel,and (totheleftinA)inA.thalianaplantsoverexpressingAtIDL1.(D)Enlarged abscissionofentiremaleinflorescence(catkin)inPopulusspp.(C)Openingof vestigialAZafterabscissionofacaulineleafinA.thalianaoverexpressing valvesindehiscencezonesofdrymany-seededcapsules,andabscissionof AtIDL1.(E,F)pHAE:GUSexpressionand(G)pHSL2:GUSexpressionin individualseeds.(D)AbscissionoffleshyfruitsatAZonpedicel.(E)Theoil vestigialAZsatthebasesofpedicels,branches,andcaulineleaves. palmdrupefruitaretightlyarrangedwithinspikeletsandabsciseonebyone whenripe.(A–D)ImagecourtesytheprivatecollectionofRoyWinkelman.First publishedinGray(1858)andFoster(1921).(E)ImagecourtesyMissouri citrus prepropeptide undergo the correct processing, that the BotanicalGarden.http://www.botanicus.org. Citrus peptide can activate IDA’s receptors and thus provides experimentalevidencesupportingafunctionincitrusabscission leaf abscission provides deciduous trees with resistance against events. drought and freezing damage (Fischer and Polle, 2010; Zanne et al., 2014). Species with N-fixing symbionts like alder (Alnus LeafAbscission spp., Fagales) can afford to lose green leaves, while other AZs, with layers of rather undifferentiated small cells, are deciduous trees, like Populus ssp. (Malpighiales), withdraw normallyformedduringorgandevelopmentbothinA.thaliana elements of valuable molecules (as chlorophyll) and shed and other species. Additionally, ethylene may promote the yellowish or reddish leaves (Keskitalo et al., 2005; Fracheboud formation of new AZs and thereby induce abscission (Roberts et al., 2009). Hence, the senescence process that allows trees to et al., 2000). A thorough investigation of IDA and HAE/HSL2 conserveresourcesandprepareforadormantperiodterminates in connection with ethylene-induced leaf abscission has been in a cell separation event in preformed AZs at the base of the performedinsoybean(Glycinemax),commonbeanandtomato pedicels. (Tucker and Yang, 2012). Of the five genes encoding tomato Leaf abscission can be induced in hybrid aspen (Populus IDA/IDL peptides, one with a PIP motif identical to that of tremula X P. tremuloides) by depriving the leaves of light (Jin mIDA was expressed in ethylene-induced AZs on leaf pedicels. et al., 2015). In our experimental setup, 50% of the shaded Likewise,theGmIDA2aand2b,whichhadthehighestexpression leaves separated from the branch at the axil after 8 days, while levelinleafAZandthehighestrelativeexpressionratiobetween non-shaded leaves were not shed. We designed gene-specific AZ and petiole tissue both in the presence and absence of primers against the IDA, IDL1, and HAE orthologs of Populus ethylene, are the paralogs most similar to AtIDA, which also ssp.,whichseemtohavelosttheHSL2ortholog.Thetwogenes display ethylene independent expression. The GmHAE3b and mostsimilartoAtIDAweresignificantlyupregulatedintheaxil GmHAE5a/5b with the highest similarities to A. thaliana HAE uponinductionofleafabscission(Figure6A).Asinsoybean,the and HSL2, respectively, were also adequately expressed in the expression levels of the receptors did not change in the course petiole(TuckerandYang,2013).Thus,theseexpressionpatterns of this experiment, suggesting that the timing of abscission is supporttheinvolvementoftheIDA-HAE/HSL2moduleinleaf ratherdependentontheinductionofIDApeptidesthanonthe abscissionbothinRosidsandAsterids. transcriptionalregulationofthereceptor. One of the most visible of all shedding processes in angiosperms is the leaf fall (Figure5B) at the beginning of the SeedandFruitAbscission dormant (i.e., cold or dry) season in deciduous trees, triggered ThefertilizedovuleofAngiosperms(theseed)andsurrounding by seasonal reduction of the photoperiod and temperature maternal tissue that together constitutes the fruit, needs to be (Taylor and Whitelaw, 2001; Keskitalo et al., 2005). Autumnal separated from the mother plant in one way or another when FrontiersinPlantScience|www.frontiersin.org 7 October2015|Volume6|Article931 Støetal. EvolutionaryconservationoftheIDApeptide FIGURE6|Continued abscisecomparedtoaxilsinnon-shaded,non-abscissionaspenleaves. qPCR,averagesandstandarddeviationsofthreebiologicalreplicates. NormalizedtoPtACTIN1expression.*p<0.05,t-test,non-shadedvs. shaded.(B)qPCRanalysisofoilpalmEgHSL1andEgHSL2expressioninAZs fromunripe(Sh1-UandSh2-U)notactivelyabscisingfruitandAZsofripe (Sh1-R,Sh3-R)activelyabscisingfruit,aswellasAZsfromripefruitofa non-abscisingtree(NSh1-R1,NSh1-R2).(C)qPCRanalysesofoilpalm EgHSLexpressionduringethylene-inducedabscissioninripe180DAPfruits. Samplesweretakenafter0,3,6,and9htreatmentwithethylene.Similar resultswereobtainedwhentreating145DAPfruits(SupplementaryFigure S3C).Inbothexperimentsfruitseparatedby9hofethylenetreatment.(D) qPCRanalysisofoilpalmEgIDA2andEgIDA5expressionintheAZsofunripe (Sh1-UandSh2-U)notactivelyabscisingfruitandAZsofripe(Sh1-R,Sh3-R) activelyabscisingfruit,aswellasAZsfromripefruitofanon-abscisingtree (NSh1-R1,NSh1-R2).ExpressionlevelsofadditionalEgIDAgenesarefoundin SupplementaryFigureS3D.(E)qPCRanalysesofoilpalmEgIDA2andEgIDA5 expressionduringethylene-inducedabscissioninripe180DAPfruits.Samples weretakenafter0,3,6,and9htreatmentwithethylene.Similarresultswere obtainedwhentreating145DAPfruits(SupplementaryFigureS3E).Inboth experimentsfruitseparatedby9hofethylenetreatment. ripe. In botanical terms there are roughly four main types of fruits: berries (fleshy most often many-seeded fruits), drupes (fleshymostoftenone-seededfruitswithhardexocarp),capsules, includingsiliquesandpods(drymany-seededfruits),andnuts, includingcaryopsesofgrasses(dryone-seededfruits).Nutsare shedandabscisedatthebaseofthecarpels,whilemany-seeded capsules or pods open along dehiscence zones (Figure5C). This is seen in a wide range of angiosperms such as orchids (Asparagales),poppies(Papaverspp.,Ranunculales),andtobacco (Nicotianatabacum,Solanales)aswellasA.thaliana,beforeeach single seed abscise where the funiculus is attached to the seed. WehaveearlierreportedthatAtIDAandHAEareexpressedin A.thalianadehiscencezones(Butenkoetal.,2006). For fleshy fruit eaten directly from the plant by birds or animals e.g., blueberries (Vaccinium myrtillus, Ericales), abscission is not necessitated for seed dispersal. Nevertheless, both larger fruits like apples (Malus domestica, Rosales) (Figure5D) and drupes like olives (Olea europaea, Asterides) (Gil-AmadoandGomez-Jimenez,2013)mayabsciseattheapex oratthebaseofthepedicel(fruitstalk),sothatanimalsmaypick upthefruitsfromtheground.Inlinewiththis,HAEwasrecently showntobestronglyupregulatedinabscisingolivescomparedto thepreabscisingAZs(Gil-AmadoandGomez-Jimenez,2013). TotestwhetherIDA-HAE/HSL2isinvolvedincontroloffruit abscission also in monocots we chose to investigate abscission of the fruits of oil palm (Elaeis guineensis, Arecales). The flowers of the oil palm grow in large clusters giving rise to plumb-sized reddish fruits in large bunches (Figure5E) that havelarge,preformed,multilayeredAZsattheboundaryofthe mesocarp and the pedicel. Under natural conditions it takes about 160 days from pollination until the fruits are ripe and start separating from the bunches on the trees. mRNA was FIGURE6|ExpressionlevelsofgenesencodingIDAligandsandHSL isolated from AZs of unripe and ripe fruit of trees that abscise receptorsinAZs.(A)qPCRdetectingincreasedexpressionlevelsofPopulus theirfruit(Shedding—Sh1,Sh2,andSh3),andadditionallyfrom IDAgenes,butnotthePtHAEgeneinaxilsofshade-treatedleavesproneto AZs of an unusual tree (Non-shedding, NSh1) that fails to (Continued) abscise its fruit. We identified genes encoding HSL1 and HSL2 FrontiersinPlantScience|www.frontiersin.org 8 October2015|Volume6|Article931 Støetal. EvolutionaryconservationoftheIDApeptide receptors; EgHSL1 had a very low expression level that did subsequent extensive loss of duplicated genes occurred in A. not change over time (Figure6B). EgHSL2 on the other hand thaliana (Tang et al., 2008; Van de Peer et al., 2009). Relevant was low in unripe AZs of shedding trees, as well as AZs of forouranalysesisthatHSL1hasbeenduplicatedintwoseparate ripe NSh1 fruit, but increased strongly in AZs of ripe fruit of events: Firstly, before the split of Vitales, Asterids, and Rosids, abscisingtrees.Fromabout145DAP,ethylenetreatmentinduce within eudicots to form HAE, and secondly, within monocots fruit abscission after 9h. This treatment also induces increased to form a more recent duplication before the split of Arecales. EgHSL2 expression (Figures6C, Supplementary Figure S3C). Still, multiple synteny investigations between five different and Thus,EgHSL2expressionwasconsistentlyassociatedwithactive partly distantly related species (Arabidopsis, Carica, Populus, fruitabscission. Vitis, and Oryza) showed that a total of 61% of the A. thaliana We identified 10 IDA oil palm genes that come in five pairs geneshadpreservedtheirancestrallocations(Tangetal.,2008). encodingalmostidenticalpreproproteins(SupplementaryFigure There are controversies regarding the placement of the smaller S3B), suggesting recent genome duplication. Expression of five clades of Ceratophyllales and Caryophyllales, as well as Vitales, of these genes (EgIDA2 to EgIDA6) was detected in AZs of E. in relation to the eudicots (Zeng et al., 2014). Our analyses guineensis fruits using RT qPCR (Supplementary Figure S3D). of the three receptor genes support Vitales and Caryophyllales IntreesshowingfruitabscissionEgIDA2,EgIDA4andthemost as sister groups of the core eudicots. HSL1/HAE duplication is abundant, EgIDA5, all had their highest expression levels when foundonsyntenicgenomeregionsintheothereudicots,butnot fruitareseparating(Figure6D),andEgIDA2andEgIDA5could rice,supportingthatHAEwasgeneratedbyaWGDevent(Tang alsobeinducedbyethylene(Figure6E).ThesegenesencodePIPs etal.,2008).SpecificgenomiclocationsofAtIDAandHSL2and neartoidenticalwithmIDA;likeCicIDA3,EgIDA5differsonly theirneighboringgenesinA.thalianaarepreservedinPopulus byanexchangeofthesmallAlawiththesmallGlyinposition6. and Vitis, respectively (Woodhouse et al., 2011). The IDL4 is Interestingly, the EgIDA genes were not strictly associated with positioned in synteny with other eudicots, but the other IDL separation; as transcripts were present also in ripe AZs of the genesmayratherbearesultofduplicationsknowntohavetaken non-sheddingtree(Figure6C).Hence,EgIDAareexpressedand placeafterthedivergenceofBrassicaceaeandCaricaceaefamilies inducedinNSh1althoughthereceptorgeneEgHSL2hasavery (Tangetal.,2008). lowlevelofexpression. Duplications provide the opportunity for the new copy to evolve, like giving rise to the PIP or PIP motif varieties. K Q DISCUSSION Transition from Arg to Lys or Gln may only require one nucleotide change. Based on available sequence data we OurphylogeneticanalysesoftheHSLLRR-RLKshaverevealed hypothesize that IDL4 with PIP evolved before the split of K that the evolution of this receptor family is congruent with Proteales, however this variant may have a more basal history that of angiosperm species evolution, and makes it likely that within angiosperm evolution. Likewise, the PIP version has Q the common ancestor of angiosperms had both a HSL1 and presumablydevelopedaftergenomeduplicationinthemonocot a HSL2 gene (Figure7). Both receptors are present already in lineage,andwehypothesizethatPIP ispresentinallmonocot Q thebasalangiosperm(e.g.,Amborella),wherestamenabscission orders (Figure7). Sequence data for Acorales is needed to is a common feature that facilitates the access of pollinators substantiate this. PIP may represent the original peptide gene R (beetles and flies bringing pollen from other flowers) to the thathasbeenlostfromsomeofthemonocotorders:Alismatales female organ (Endress, 2010). The IDA gene with PIP motif and Poales are devoid of the gene, whilst it is present in R identifiedinAmborella,mayplayarolehere,similartoIDA’srole ArecalesandZingiberales,suggestingindependentlossforsome asasignalingligandinsepal,petal,andstamenabscissioninA. lineages.Thisismoreparsimoniousthanabasallosswithinthe thaliana(Butenkoetal.,2003,2014). monocots followed by multiple gains. A final conclusion must While the receptors follow the general evolution of the awaitgenomicsequencingofspeciesfromorderswheredataat angiosperms, the PIP motif likely to represent the peptide presentarelacking. R releasedfromthepropeptideuponsecretion,hasvirtuallybeen We have evidence from A. thaliana that IDA can be preservedwithoutsubstantialchangesthroughouttheevolution substituted with AtIDL1, but the other AtIDL genes only to of flowering plants. In a short peptide that presumably shall a limited extent can substitute for IDA function, indicating bind to given aa side chains in the receptor, there are limited that their encoded peptides do not interact properly with the possibilities for aa changes that retain the relevant binding HAE and HSL2 receptors (Stenvik et al., 2008). The high properties:Exchangeofonesmallaaforanothermightpass,but similarity between conserved aa in the ectodomain as well as exchangeofanaawithdifferentsizeorchargeofthesidechain the kinase domain of HSL1 compared to HAE, might suggest (likeaLysoraAsnforanArg),mayaffectthestructureofthe HSL1 to be their native receptor. This would require closely ligandanditsbindingproperties(Czyzewiczetal.,2015). related expression patterns for HSL1 and IDL genes. However, The genome of a common ancestor of flowering plants has at least in Arabidopsis, the expression patterns of HSL1 and been suggested to harbor between 10,000 and 14,000 genes these IDL genes do not suggest involvement in floral organ (Tang et al., 2008) while a genome size of 25,000–50,000 genes abscission (Cho et al., 2008; Stenvik et al., 2008), and mutant is common in modern angiosperms. This increase is due to phenotypes are so far lacking. Different methods have lately a number of WGD, including a triplication in dicots before beensuggestedfortestingofpeptide-ligandreceptorinteractions the separation of the Vitis. Two additional duplications and a (Butenko et al., 2009, 2012, 2014). Interaction of the mIDA FrontiersinPlantScience|www.frontiersin.org 9 October2015|Volume6|Article931 Støetal. EvolutionaryconservationoftheIDApeptide FIGURE7|EvolutionoftheIDAHAE/HSL2signalingmodule.PhylogenyofAngiospermsadaptedfromZhangetal.(2012),Vannesteetal.(2014),Zengetal. (2014),andDohmetal.(2014).TaxonomicallevelsaretakenfromZengetal.(2014).Numbersinsuperscriptbehindordernamesrepresentthenumberofspecies usedintheanalysis.Ordernamesinboldrepresentthoseorderswithatleastonecompletelysequencedgenome.ThetwopossibleevolutionaryoriginsofIDA(PIPQ) inmonocotsareillustrated. peptide with HSL2 expressed in N. benthamiana, results in an (diminished in the domesticated species) in the grasses. We immediateoxidativeburst,similartoelicitedLRR-RLKsinvolved have demonstrated that oil palm of the Arecales use HSL2 in pathogen defense (Butenko et al., 2014). A thousand times and IDA genes encoding PIP peptides in connection to fruit R morepeptideisneededtoprovokesuchareactionfromtheHAE abscission. It is tempting to suggest that the PIP version of Q receptorunderthesameconditions,althoughthetworeceptors the IDA peptide have developed to fit one or the other HSL1 in genetic terms are functionally redundant with respect to receptors. control of floral organ abscission (Cho et al., 2008; Butenko We have presented examples of the presence of IDA and et al., 2014). We have found distinct evolutionarily conserved its receptors in all the major groups of Angiosperms during differencesbetweenthekinasedomainsofHAE/HSL1andthatof abscissionoffloralorgans,flowers,immature,andmaturefruits HSL2.ThismayexplainthedifferencesinreadoutbetweenHAE and of leaves. The first Angiosperm developed more than 150 andHSL2inthepresenceofIDAorIDLpeptides(Butenkoetal., Myrago,andIDAligandsmayhavebeenconservedmostofthis 2014). time. At the same time the number of IDA and receptor genes Interestingly, within monocots HSL1 has been duplicated has increased and a variety of modes of organ detachment has beforethesplit-offoftheArecales,andHSL2hasbeenlostafter developed. This lends support to the notion that it is not what thesplitofArecales,leavingtheordersPoales,andZingiberales youdo,butwhereandwhenyoudoitthatisimportant(Carroll, devoid of the gene. Abscission still takes place in these orders, 2008). New gene copies have presumably made it possible to e.g.,“fingerdrop”inbanana(Musaspp.)andcaryopsisshedding changeplaceandtimeforcellseparationevents. FrontiersinPlantScience|www.frontiersin.org 10 October2015|Volume6|Article931