Characterization of the basal angiosperm Aristolochia fimbriata

26 Pages·2013·5.85 MB·English

Characterization of the basal angiosperm
Aristolochia fimbriata: a potential experimental
system for genetic studies
Bliss et al.
Blissetal.BMCPlantBiology2013,13:13
http://www.biomedcentral.com/1471-2229/13/13
Blissetal.BMCPlantBiology2013,13:13
http://www.biomedcentral.com/1471-2229/13/13
RESEARCH ARTICLE Open Access
Characterization of the basal angiosperm
Aristolochia fimbriata: a potential experimental
system for genetic studies
Barbara J Bliss1,2*†, Stefan Wanke3†, Abdelali Barakat1,4, Saravanaraj Ayyampalayam5, Norman Wickett1,6,
P Kerr Wall1,7, Yuannian Jiao1, Lena Landherr1, Paula E Ralph1, Yi Hu1, Christoph Neinhuis3, Jim Leebens-Mack5,
Kathiravetpilla Arumuganathan8, Sandra W Clifton9,10, Siela N Maximova11, Hong Ma1,12,13
and Claude W dePamphilis1*
Abstract
Background: Previous studies inbasal angiospermshave provided insight intothe diversity within the angiosperm
lineage and helped to polarize analysesof flowering plant evolution. However, there is still notan experimental
system for genetic studies among basal angiosperms to facilitate comparative studies and functional investigation.
It would be desirable to identify a basal angiosperm experimental system that possesses many of the features
found inexisting plant model systems (e.g., Arabidopsisand Oryza).
Results: We have considered all basal angiosperm families for general characteristics important for experimental
systems, including availability to thescientific community, growth habit, and membership in a large basal
angiosperm group that displays a wide spectrum ofphenotypicdiversity.Most basalangiospermsare woody or
aquatic, thus are notwell-suited for large scale cultivation, and were excluded. We further investigated members of
Aristolochiaceae for ease of culture, life cycle, genome size, and chromosome number. We demonstrated
self-compatibility for Aristolochia elegans and A. fimbriata, and transformation witha GFP reporter construct for
Saruma henryi and A. fimbriata. Furthermore, A. fimbriata was easily cultivated with a life cycle of just three months,
could be regenerated ina tissue culture system, and had one ofthe smallest genomes among basal angiosperms.
Anextensive multi-tissue EST dataset was producedfor A. fimbriata that includes over3.8 million 454 sequence
reads.
Conclusions: Aristolochia fimbriata has numerous features that facilitategenetic studies and is suggestedas a
potential model system for use with a wide variety of technologies. Emerging genetic and genomic tools forA.
fimbriata and closely related species can aid the investigation offloral biology,developmental genetics,biochemical
pathways important inplant-insect interactions as well as humanhealth,and various other features present inearly
angiosperms.
*Correspondence:[email protected];[email protected]
†Equalcontributors
1DepartmentofBiology,InstituteofMolecularEvolutionaryGenetics,andthe
HuckInstitutesoftheLifeSciences,201LifeSciencesBuilding,Pennsylvania
StateUniversity,UniversityPark,PA16802,USA
2USDAARSPBARC,64NoweloSt.,Hilo,HI96720,USA
Fulllistofauthorinformationisavailableattheendofthearticle
©2013Blissetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative
CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginalworkisproperlycited.
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Table1Asummaryofrelevantbasalangiospermcharacteristics
Order Family1,2 #Genera/species3 Familiargenera3 Growthform1,2 Commerciallyavailable
AMBORELLALES Amborellaceae 1/1 Amborella shrub no
NYMPHAEALES Nymphaeaceae 5/58 Nymphaea aquaticherb yes
Nuphar
Barclaya
Victoria
Euryale
Cabombaceae 2/6 Cabomba aquaticherb yes
Brassenia
Hydatellaceae 2/10 Hydatella aquaticherb no
Trithuria
AUSTROBAILEYALES Austrobaileyaceae 1/2 Austrobaileya liana no
Illiciaceae 3/92 Illicium shrub,tree yes
Schisandra
Kadsura
Trimeniaceae 1-2/6 Trimenia shrub,tree,liana no
CERATOPHYLLALES Ceratophyllaceae 1/6 Ceratophyllum aquaticherb yes
CHLORANTHALES Chloranthaceae 4/75 Chloranthus herb,shrub,tree no
Ascarina
Hedyosmum
Sarcandra
MAGNOLIALES Annonaceae 129/2220 Annona shrub,tree,liana yes
Guatteris
Xylopia
Uvaria
Polyalthia
Rollinia
Artabotrys
Asimina
Eupomatiaceae 1/3 Eupomatia shrub,tree yes
Magnoliaceae 2/227 Magnolia shrub,tree yes
Lioriodendron
Degeneriaceae 1/2 Degeneria tree no
Himantandraceae 1/2 Galbulimima tree no
Myristicaceae 20/475 Myristica shrub,tree yes
Horsfieldia
Virola
Knema
LAURALES Calycanthaceae 5/11 Chimonanthus shrub,tree yes
Calycanthus
Idiospermum
Sinocalycanthus
Hernandiaceae 5/55 Hernandia shrub,tree,liana no
Illigera
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Table1Asummaryofrelevantbasalangiospermcharacteristics(Continued)
Lauraceae 50/200 Laurus shrub,tree,parasiticvine yes
Litsea
Ocotea
Cinnamomum
Cryptocarya
Persea
Lindera
Neolitsea
Nectandra
Phoebe
Apollonias
Beilschmiedia
Umbellularia
Monimiaceae 22/200 Doryphora shrub,liana no
Peumus
Xymalos
Mollinedia
Tambourissa
Kibara
Siparunaceae 2/75 Siparuna shrub,tree no
Glossocalyx
Gomortegaceae 1/1 Gomortega shrub,tree no
Atherosperma-taceae 6-7/16 Atherosperma shrub,tree no
Daphnandra
Doryphora
Dryadodaphne
Laurelia
Nemuaron
CANELLALES Canellaceae 5/13 Canella shrub,tree yes
Cinnamodendron
Cinnamosma
Winteraceae 4-7/60-90 Drimys shrub,tree yes
Zygogynum
Pseudowintera
Takhtajania
PIPERALES Hydnoraceae 2/7 Prosopanche parasiticherb no
Hydnora
Piperaceae 5/3600 Peperomia herb yes
Piper herb yes
Zippelia herb no
Manekia liana no
Verhuellia herb no
Saururaceae 5/6 Anemopsis herb yes
Houttuynia herb yes
Saururus herb yes
Gymnotheca herb no
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Table1Asummaryofrelevantbasalangiospermcharacteristics(Continued)
Lactoridaceae 1/1 Lactoris shrub no
Aristolochiaceae 4/550 Saruma(1) herb yes
Asarum(ca.86) herb yes
Thottea(ca.29) shrub no
Aristolochia(ca.450) herb,shrub,liana yes
Familiargeneraincluderepresentativesfromthefamilyandarenotintendedtocompriseacomprehensivelisting.Taxaareconsidered“commerciallyavailable”if
onehundredplantsormorecanbepurchased,inexpensively,andcanbereadilypropagatedfromseed.
1[9].
2[17].
3NumberofspeciesshownforAristolochiaceaegenerafrom[18].
Background Illiciaceae,Trimeniaceae, and Austrobaileyaceae), followed
Our present understanding of genetics, genomics, devel- by the magnoliids (Figure 1). The genome of the shrub
opment, evolution, and physiology of living organisms Amborella trichopoda, the likely singular living sister
has benefited greatly from work done in model genetic group to the rest of the living angiosperms, has been
systems. Model systems provide more favorable condi- mapped[10]andsequenced(www.Amborella.org),provid-
tions for observing phenomena and testing hypotheses ing an ideal root for comparative genomic studies of all
than othersystemsafford.Modelssupportinductive rea- other angiosperms [11]. The magnoliids contain four
soning, in which one builds on the understanding of liv- major branches and several thousand species (Table 1,
ing organisms in general, based on observations made in Figure 1). Among the species in this lineage are the orna-
a specific model organism [1]. Model organisms also mentaltree,Liriodendrontulipfera[12]and the fruit tree,
provide a focus for researchers to work on a common Persea americana [13], as well as the economically
system, resulting in collaborative and complementary important spice, black pepper (Piper nigrum) [14].
efforts that can yield rapid progress and development of Taxa included in the magnoliids exhibit many features
further resources. Models are crucial for understanding first appearing in basal angiosperms or angiosperms in
basicbiologicalprocesses. general e.g., vessel elements, perianth bilateral symmetry,
Model organisms should have several key attributes. alkaloidchemistry,specializedpollinationsystems,anddi-
They should share a number of characteristics of the verseformsoffemalegametophytedevelopment[3,15,16].
taxon or process they are chosen to represent [1,2] and Recent phylogenetic studies have suggested that the mag-
must be accessible [2] so that a broad community of noliids are the sister clade and therewith the closest out-
scientists can utilize and develop them. Models used for group to the species-rich and highly diverged monocot
developmental and genetic studies must also offer rapid andeudicotlineages[7-9].
development, short generation time, be amenable to The rich diversity of basal angiosperms provides
large scale cultivation, have small seed size for easy stor- glimpses into early successful experiments in angio-
age of many genotypes, and provide ample tissue for ex- sperm adaptation [3,19-23]. Novel features which are
perimentation [1]. Models should also support forward otherwise constrained by function may have evolved
andreverse genetics,asisrequiredfor hypothesis testing more than once, through parallelism and convergence,
[3], and have a small genome size to facilitate molecular such as the structural and developmental similarities of
genetics and genomics work, including genome sequen- the inflorescence of the monocot Acorus with that found
cing and assembly [4,5]. Finally, for studying the evolu- in some Piperales (Piperaceae, Saururaceae; members of
tion of development, models should have both Magnoliids) [24]. Basal lineages also retain evidence of
conserved and unique features in comparison to related “trials” of features that became genetically fixed in later
species, so that comparative studies can elucidate the lineages [25]. For example, perianth parts are quite vari-
mechanisms ofphenotypic evolution[6]. able in basal angiosperms, as well as in basal eudicots
Ourcurrent understandingof angiospermevolution has and basal monocots [26], and only became canalized
beenshapedbymultiplephylogeneticstudieseg.,[7-9]that later on.
provide the organismal context in which the evolution of Current flowering plant genetic models are all derived
any aspect of flowering plants is studied. Of particular from the highly diverged monocot and eudicot lineages
interest to both basic and applied plant biology are (Figure 1). Among them, the monocot models Zea and
changesleadingtothesuccessanddiversificationofangio- Oryza occur in Poaceae, which has specialized floral
sperm lineages, beginning with the early, mostly species- organs and an inflorescence found only in that family.
poor lineages of angiosperms (Table 1) previously known Similarly, the current eudicot models are derived from
as the ANITA grade (Amborellaceae, Nymphaeales, the rosid (Arabidopsis) and asterid (e.g., Antirrhinum,
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Amborellales
Nymphaeales
Austrobaileyales
Ceratophyllales
Chloranthales
Magnoliales Liriodendron (tree)
Laurales
Persea(tree)
Canellales
Piperales Aristolochia
Acorales
magnoliids Alismatales
Petrosaviales
Dioscoreales
Pandanales
Liliales
Asparagales Asparagus
Arecales Oryza*
basal Poales Zea*
angiosperms monocots Commelinales Brachypodium
Zingiberales
Aquilegia*
Ranunculales
Sabiales Eschscholzia*
Proteales
Trochodendrales
Buxales
Gunnerales
Berberidopsidales
Dilleniales Beta
Caryophyllales Mesembryanthemum*
Santalales
Saxifragales
Vitales Vitis
Crossosomatales
Geraniales
Myrtales
Zygophyllales
Celastrales
Malpighiales Populus(tree)
Oxalidales
eudicots Fabales Medicago
Rosales Glycine
rosids
Cucurbitales Pisum
Fagales Lotus*
Huerteales Arabidopsis*
Brassicales Brassica
Malvales Carica(tree)
Sapindales
Gossypium
Cornales
Ericales
Garryales
Antirrhinum*
Gentianales
Mimulus
asterids Lamiales
Solanales Solanum
Aquifoliales Lycopersicon
Apiales Nicotiana
Asterales Petunia*
Dipsacales Helianthus
Gerbera*
Figure1AngiospermphylogenybasedonStevens[9],modified(http://www.mobot.org/MOBOT/research/APweb/welcome.html).
Importantmodelsystemsandtheproposedmodel,Aristolochiafimbriata,areshownnexttothecorrespondingclade.Liriodendron,Persea,
Populus,andCaricaaretreespecies.Speciesthathavebeenusedasflowerdevelopmentmodelsareindicatedwithanasterisk.
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tomato) “core eudicot” lineages, each of which displays commercially at a low price and could be readily pro-
lineage-specific floral forms [25,27]. Studies in current pagated by seed. Those of limited availability were
plantmodelshaveledtothediscoveryofbroadlyhomolo- eliminated, including Amborellales (Amborellaceae),
gous traits, including conservation of floral organ identity Austrobaileyales (Illiciaceae, Austrobaileyaceae, Trime-
genes (ABC/quartet models) [28-30]. These homologies, niaceae), andChloranthales (Chloranthaceae) (Figure 2).
and others observed in current plant models (Figure 1), Next, we eliminated groups of plants sharing a growth
havesuggestedhypothesesaboutthecommonancestorof habit associated with barriers for use as a genetic model.
monocotsandeudicots. Woody plants requiring a year or more to attain maturity,
An understanding of the evolution of floral develop- as well as extensive space for cultivation, were elim-
ment or any fundamental process in flowering plants inated(Figure2).TheseincludedmostMagnoliales(Myris-
should include results from a basal angiosperm experi- ticaceae, Magnoliaceae, Annonaceae, Himantandraceae,
mental model in which functional hypotheses can be Degeneriaceae, Eupomatiaceae), Laurales (Lauraceae, Her-
tested [3,6,31-33]. Although the current model systems nandiaceae, Monimiaceae, Atherospermataceae, Gomorte-
represent well the highly successful and derived lineages gaceae, Siparunaceae, Calycanthaceae), and Cannellales
inwhichtheyoccur,theydonotrepresenttheoveralldi- (Cannellaceae, Winteraceae). The forest tree species, Lirio-
versity of angiosperms. Information from basal lineages dendron (Magnoliaceae) and the fruit tree Persea (Laura-
is necessary to better describe that diversity, to polarize ceae), were among those eliminated due to woody habit
the changes that occurred during angiosperm evolution, andlonggenerationtime.Manyofthefamilieslistedabove
and to make functional inferences about the common would also be eliminated due to limited commercial
ancestor ofearlyangiosperms. availability, as e.g., Trimeniaceae, Himantandraceae, and
Currentplantmodelshavebeenselectedtoaddresspar- Gomortegaceae (Table 1). Similarly, many of the families
ticularquestions,butveryfewareavailableforusebothas eliminated for limited accessibility have a woody habit
genomic and as genetic models. Genomic resources, (Table1);thesewouldhavebeeneliminatedforthatcharac-
which emphasize plants with relatively small genomes, ter even if they had been more accessible (e.g., Amborella
have been developed for tree wood and fruit species, in- trichopoda). Plants with an aquatic habit, including water
cluding Populus [34], Liriodendron [12], Persea [35] and lilies(Nymphaeales:Nymphaceae,Cabombaceae)andCera-
Carica [36]. However, woody species are too large at ma- tophyllum (Ceratophyllales: Ceratophyllaceae), were elimi-
turityanddonothaveshortenoughlifecyclesforgeneral nated due to the extensive cultivation requirements
use in genetic experiments. Forward genetics requires a associated with maintaining large numbers of individual
very small organism with a rapid life cycle, and is facili- aquatic plants, as well as the difficulty in developing self-
tated by the ability to self-pollinate individuals having pollination and transformation protocols in an aquatic en-
desired characteristics. Reverse genetics requires manipu- vironment (Figure 2). Orders composed entirely of woody
lationofDNAorRNAinatargetedmanner.Bothbenefit oraquaticplantswereeliminatedafterreviewandconsider-
from a small genome, and transformability is essential for ationofthespeciescomprisingthem.
testing hypotheses about gene function. Therefore, we The order Piperales (Aristolochiaceae, Hydnoraceae,
sought to identify a basal angiosperm species having as Piperaceae, Saururaceae, Lactoridaceae) contains several
manyimportant featuresof a model systemas possible to herbaceous taxa (Table 1). Parasitic plants (Hydnoraceae)
support its potential development into an experimental andthosewithhighlyreducedflowers(Saururaceae,Piper-
systemingeneticsandgenomics.Wepresenttheseessen- aceae)donotgenerallyrepresentangiosperms,andsowere
tial features in Aristolochia fimbriata - small size at ma- eliminated (Figure 2). Hydnoraceae and Lactoridaceae
turity, rapid life cycling, self-compatibilty, small genome wouldalsobeexcludedduetolimitedavailability(Table1).
size, and transformability - along with relevant findings Among the basal angiosperm families, only Aristolochia-
forothertaxaevaluatedinourstudy. ceaecontainshighlyaccessible,easilycultivatedherbaceous
plants with features broadly representative of angiosperms
Results ingeneral.
Evaluationofpotentialmodelsinbasalangiosperm
ordersandfamilies Aristolochiaceaecandidatesconsidered
Wefollowedaformalselectionprocesstoidentifyasuit- We surveyed Aristolochiaceae, seeking species with
able candidate for an experimental organism among rapidgrowth,norequirementfor vernalizationinthelife
basal angiosperms (Figure 2). Many basal angiosperms cycle, ease of large scale cultivation, and a small genome
are uncommon, with limited distribution, and often size to facilitate gene function studies and genome se-
occur in families with only one genus and few species quencing and assembly (Figure 2). Members of genus
(Table 1). Taxa were considered readily accessible to a Aristolochia have some of the smallest basal angiosperm
broad scientific community if they could be obtained genome sizes currently known (Figure 3). Therefore, we
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Figure2Overallapproachforselectingabasalangiospermmodelsystem.Generalcriteriaareindicated;forfulldescriptionreferto
methods.Taxaeliminatedafterinitialapplicationofeachcriterionareindicated.Sometaxamayhavebeeneliminatedformorethanonereason.
Forexample,Illicium,infamilyIlliciaceae,isincreasinglyavailableincultivation,unlikemostAustrobaileyales,butwaseliminatedduetoitswoody
growthhabit.ManytaxainPiperalesweregenerallyaccessibleandofamenablegrowthhabit,yetfamilyLactoridaceaewaseliminateddueto
inaccessibilityandwoodiness.IngenusAristolochia,subgenusPararistolochiawaseliminatedduetolargegenomesize.Basalangiospermfamily
characteristicsandthoseofAristolochiaceaespeciescultivatedaredescribedinTables1and2.
evaluated each genus in this family to identify the best so they were eliminated (Figure 2). Thottea, here repre-
species for model system development. Subfamily Asar- sented withT. siliquosa (Figure 4C), was not possible to
oideae genera (Asarum, Saruma) (Figure 4A and B) re- obtain for a detailed cultural survey (Table 2), as it can
quire cold treatment to induce flowering, resulting in only be cultivated with very high maintenance, under a
increased culturing efforts and extended time to flower, narrow range of conditions. Furthermore, it grows
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Figure3Genomesizesinbasalangiospermfamilies.BennettandLeitch[38]updatedwithCuietal.[39],shownonlogarithmicscale.Filled
symbolsindicatetaxausedinTheFloralGenomeProject(www.floralgenome.org)orTheAncestralAngiospermGenomeProject(http://ancangio.
uga.edu/),andforwhichESTresourcesareavailable.ComparebasalangiospermgenomesizestoArabidopsisat125Mb[40]andOryzaat389
Mb[41].ThesymbolrepresentingAristolochiaistheproposedmodelAristolochiafimbriata.OtherspeciesofAristolochiahavesmallergenome
sizes(seeFigure5,Table4).
slowly, produces little tissue, few flowers, and few seeds. smallest genome size to date from a basal angiosperm
Of the Aristolochia species available for culturing, those (A. lindneri) (Figure 5). In order to gain insight into the
requiring cold treatment (e.g. A. serpentaria, other evolution of genome structure as well as genome size in
members of subgenus Isotrema and A. clematitis, Aristolochiaceae, chromosome numbers from previously
Figure 4D) were eliminated from consideration. Mem- published studies [43,44] were plotted along with gen-
bers of subgenus Pararistolochia (Figure 4E) were not ome sizes on a strict consensus tree using TreeGraph
available for culturing or are large vines with a long life [45] (Figure 5, see also Additional file 1: Phylogram of
cycle. Those species that did not bloom in six months Aristolochiaceaerelationships).
(A. californica, A. anguicida, A. macrophylla, A. tomen- Adirectcorrelationbetweenchromosomenumbersand
tosa), produced very few flowers (A. trilobata, A. passi- genome size for the family of Aristolochiaceae was not
florafolia; Figure 4 F and G), or formed very large vines observed.Aristolochiaceaeissubdividedintotwosubfam-
(A. grandiflora, A. ringens, A. labiata, A. gigantea) ilies, Asaroideaeand Aristolochioideae.Asaroideae,which
(Table 2, Figure 2) were also eliminated. The remaining has2n= 26,52(Sarumahenryi)and 2n=26(Asarumcau-
candidates that met our criteria were two smaller mem- datum) chromosomes, has about two to ten times the
bers of subgenus Aristolochia (A. elegans and A. fim- genomesizeofgenusAristolochia(Figure5).Thottea,the
briata), belonging to a group of subtropical and tropical earliest diverging branch in subfamily Aristolochioideae
species from South andCentralAmerica[18,37]. hasthesamenumberofchromosomes(2n=26)asAsarum
andSaruma,buthasonly1/9and1/5ofthegenomesize
Aphylogeneticperspectiveofgenomesizes ofAsarumandSaruma,respectively.
Because a small genome size facilitates molecular and In contrast, within genus Aristolochia, species in sub-
functional investigation of genes of interest, including genus Aristolochia exhibitthe smallest genome sizes,but
cloning and characterization of both coding and regula- have a wide range of chromosome numbers. Species in
tory regions, and genome sequencing and assembly, our subgenus Isotrema (Figure 4D) are generally character-
analysis of genome size evolution in Aristolochiaceae fo- ized by 2n=32 chromosomes and have small genome
cused on species having small genomes, particularly in sizes (554-774Mbp meangenomesize, 1C). Within sub-
subgenus Aristolochia, for which we report here the genus Pararistolochia(Figure 4E) (1793-4321 Mbp mean
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Figure4DiversityofflowerandgrowthformsinAristolochiaceae.Herbaceousperennialswithradiallysymmetric3-merousflowersinclude
A.AsarumchingchengenseB.SarumahenryiC.Thotteasiliquosa,asmallshrubD.A.arborea(subgenusIsotrema),atree-likeshrubwithflowers
thatmimicfungiE.A.triactina(subgenusPararistolochia)F.A.trilobata(subgenusAristolochia)withthreelobed,evergreenleaves,growsasavine
withwoodybranches(liana)fromwhichnewgrowthemerges.G.A.passiflorafolia(subgenusAristolochia)(photousedwithpermissionfrom
ChangbinChen)andH.A.fimbriata(subgenusAristolochia).
genome size, 1C), which is sister group to subgenus previous studies [14,18]. It is interesting to note that
Aristolochia, and therefore nested within the clade Asarum and subgenus Pararistolochia have large chro-
having species with small genomes, a lineage-specific mosomes similar to those of monocots, whereas the
increase in genome size can be seen. The increase is not remainingcladesinAristolochiaceaehavesmallchromo-
associated with an increase in chromosome number, somes [43]. Aristolochia fimbriata is a member of a
but in chromosome size [43]. The Australian species clade of Aristolochia species with 2n=14 chromosomes
of Pararistolochia have less than half of the genome size andgenomesroughlythesizeofOryza sativa.
of the African species, but still have the same number of
chromosomes [43]. Within the subgenus Aristolochia Methodsforgenetics
(Figure 4F, G and H), the different monophyletic groups We further evaluated selected taxa for self-compatibility
recovered(Figure5;seealsoAdditionalfile1:Phylogram and potential for genetic engineering, both of which are
of Aristolochiaceae relationships) are in accordance with critical features of genetic systems. Self-pollination