Insights into the red algae and eukaryotic evolution from the genome of Porphyra umbilicalis

124 Pages·2017·12.07 MB·English

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Insights into the red algae and eukaryotic evolution P
Porphyra umbilicalis
from the genome of
(Bangiophyceae, Rhodophyta)
SusanH.Brawleya,1,Nicolas A.Blouina,b,ElizabethFicko-Bleanc,GlenL.Wheelerd,MartinLohre,Holly V.Goodsonf,
JerryW.Jenkinsg,h,CrystenE.Blaby-Haasi,KatherineE. Helliwelld,j,CheongXinChank,l,TaraN.Marriagem,
DebashishBhattacharyan,AnitaS.Kleino,YacineBadisp,JulietBrodieq,YuanyuCaoo,2,JonasCollénc,SimonM.Dittamic,
ClaireM. M.Gachonp,BeverleyR.Greenr,StevenJ.Karpowiczs,JayW.Kimt,UlrichJohanKudahlj,SenjieLinu,
GurvanMichelc,MariaMittagv,BradleyJ.S.C.Olsonm,JasmynL.Pangilinanh,YiPengh,HuanQiun,ShengqiangShuh,
JohnT.Singerw,AlisonG.Smithj,BrittanyN.Sprecheru,VolkerWagnerv,WenfeiWangx,Zhi-YongWangy,JuyingYanh,
CharlesYarishz,SimoneZäuner-Riekaa,YunyunZhuangu,3,YongZouv,ErikaA.Lindquisth,JaneGrimwoodg,h,
KerrieW.Barryh,DanielS.Rokhsarh,JeremySchmutzg,h,JohnW.Stillerbb,ArthurR.Grossmany,andSimonE.Prochnikh
aSchoolofMarineSciences,UniversityofMaine,Orono,ME04469;bDepartmentofMolecularBiology,UniversityofWyoming,Laramie,WY82071;
cSorbonneUniversités,UniversitéPierreandMarieCurieParis06,CNRS,UMR8227,IntegrativeBiologyofMarineModels,StationBiologiquedeRoscoff,CS
90074,29688Roscoff,France;dMarineBiologicalAssociationoftheUnitedKingdom,Plymouth,PL12PB,UnitedKingdom;eInstitutfürMolekulare
Physiologie,Pflanzenbiochemie,JohannesGutenberg-UniversitätMainz,55128Mainz,Germany;fDepartmentofChemistry&Biochemistry,Universityof
NotreDame,SouthBend,IN46556;gHudsonAlphaInstituteforBiotechnology,Huntsville,AL35806;hDepartmentofEnergy,JointGenomeInstitute,
WalnutCreek,CA94598;iBiologyDepartment,BrookhavenNationalLaboratory,Upton,NY11973;jDepartmentofPlantSciences,UniversityofCambridge,
CambridgeCB23EA,UnitedKingdom;kInstituteforMolecularBioscience,UniversityofQueensland,Brisbane,QLD4072,Australia;lSchoolofChemistry
andMolecularBiosciences,UniversityofQueensland,Brisbane,QLD4072,Australia;mDivisionofBiology,KansasStateUniversity,Manhattan,KS66506;
nDepartmentofEcology,Evolution&NaturalResources,RutgersUniversity,NewBrunswick,NJ08901;oDepartmentofBiologicalSciences,Universityof N
NewHampshire,Durham,NH03824;pScottishAssociationforMarineSciences,ScottishMarineInstitute,ObanPA371QA,UnitedKingdom;qNatural TIO
HistoryMuseum,DepartmentofLifeSciences,LondonSW75BD,UnitedKingdom;rDepartmentofBotany,UniversityofBritishColumbia,VancouverBC, LU
CanadaV6T1Z4;sTauBiosciencesLLC,Edmond,OK73003;tDepartmentofBiomolecularEngineering,UniversityofCalifornia,SantaCruz,CA95064; VO
uDepartmentofMarineSciences,UniversityofConnecticut,Groton,CT06340;vInstitutfürAllgemeineBotanikundPflanzenphysiologie,Friedrich-Schiller- E
UniversitätJena,07743Jena,Germany;wDepartmentofMolecular&BiomedicalScience,UniversityofMaine,Orono,ME04469;xBasicForestryand
ProteomicsResearchCenter,HIST,FujianAgricultureandForestryUniversity,Fuzhou350002,China;yDepartmentofPlantScience,CarnegieInstitutionfor
Science,Stanford,CA94305;zDepartmentofEcology&EvolutionaryBiology,UniversityofConnecticut,Stamford,CT06901;aaInstituteofMolecular
PhysiologyandBiotechnology,UniversityofBonn,53115Bonn,Germany;andbbDepartmentofBiology,EastCarolinaUniversity,Greenville,NC27858
EditedbyStephenR.Palumbi,StanfordUniversity,PacificGrove,CA,andapprovedJune6,2017(receivedforreviewFebruary22,2017)
Porphyra umbilicalis (laver) belongs to an ancient group of red haptophytes have strong biogeochemical effects; apicomplexans
algae(Bangiophyceae),isharvestedforhumanfood,andthrives causehumandisease(e.g.,malaria);anddinoflagellates include
intheharshconditionsoftheupperintertidalzone.Herewepre- both coral symbionts and toxin-producing “red tides” (1). The
sent the 87.7-Mbp haploid Porphyra genome (65.8% G + C con- evolutionary processes that produced the Archaeplastida and
tent, 13,125 gene loci) and elucidate traits that inform our secondaryalgallineagesremainunderinvestigation(2–5),butit
understandingofthebiologyofredalgaeasoneofthefewmul- is clear that both nuclear and plastid genes from the ancestral
ticellulareukaryoticlineages. Novel featuresof thePorphyrage- red algae have contributed dramatically to broader eukaryotic
nomesharedbyotherredalgaerelatetothecytoskeleton,calcium evolution and diversity. Consequently, the imprint of red algal
signaling,thecellcycle,andstress-tolerancemechanismsincluding metabolismontheEarth’sclimatesystem,aquaticfoodwebs,and
photoprotection.Cytoskeletalmotorproteinsin Porphyraarere-
strictedtoasmallsetofkinesinsthatappeartobetheonlyuni-
versalcytoskeletalmotorswithintheredalgae.Dyneinmotorsare Authorcontributions:S.H.B.,N.A.B.,D.B.,S.L.,C.Y.,K.W.B.,D.S.R.,J.S.,J.W.S.,A.R.G.,and
absent,andmostredalgae,includingPorphyra,lackmyosin.This S.E.P.designedresearch;S.H.B.,N.A.B.,E.F.-B.,G.L.W.,M.L.,H.V.G.,J.W.J.,C.E.B.-H.,
K.E.H.,C.X.C.,T.N.M.,D.B.,A.S.K.,Y.B.,J.B.,Y.C.,J.C.,S.M.D.,C.M.M.G.,B.R.G.,S.J.K.,
surprisingly minimal cytoskeleton offers a potential explanation J.W.K.,U.J.K.,S.L.,G.M.,M.M.,B.J.S.C.O.,J.L.P.,Y.P.,H.Q.,S.S.,A.G.S.,B.N.S.,V.W.,
forwhyredalgalcellsandmulticellularstructuresaremorelimited W.W.,Z.-Y.W.,J.Y.,S.Z.-R.,Y.Zou,E.A.L.,J.G.,K.W.B.,D.S.R.,J.S.,J.W.S.,A.R.G.,and
insizethaninmostmulticellularlineages.Additionaldiscoveries S.E.P.performedresearch;S.H.B.,N.A.B.,E.F.-B.,G.L.W.,M.L.,H.V.G.,J.W.J.,C.E.B.-H.,
K.E.H.,C.X.C.,D.B.,S.M.D.,C.M.M.G.,S.J.K.,G.M.,H.Q.,A.G.S.,J.S.,J.W.S.,andS.E.P.
further relating to the stress tolerance of bangiophytes include
contributednewreagents/analytictools;S.H.B.,N.A.B.,E.F.-B.,G.L.W.,M.L.,H.V.G.,
ancestral enzymes for sulfation of the hydrophilic galactan-rich J.W.J.,C.E.B.-H.,K.E.H.,C.X.C.,T.N.M.,D.B.,A.S.K.,Y.B.,J.B.,Y.C.,J.C.,S.M.D.,C.M.M.G.,
cell wall, evidence for mannan synthesis that originated before B.R.G.,S.J.K.,J.W.K.,U.J.K.,S.L.,G.M.,M.M.,B.J.S.C.O.,H.Q.,A.G.S.,B.N.S.,V.W.,W.W.,
the divergence of green and red algae, and a high capacity for Z.-Y.W.,S.Z.-R.,Y.Zou,J.S.,J.W.S.,A.R.G.,andS.E.P.analyzeddata;S.H.B.,N.A.B.,S.L.,J.T.S.,
C.Y.,Y.Zhuang,andA.R.G.preparedsamples;andS.H.B.,N.A.B.,E.F.-B.,G.L.W.,M.L.,H.V.G.,
nutrient uptake. Our analyses provide a comprehensive under-
J.W.J.,C.E.B.-H.,K.E.H.,C.X.C.,T.N.M.,D.B.,A.S.K.,Y.B.,J.B.,Y.C.,J.C.,S.M.D.,C.M.M.G.,B.R.G.,
standingoftheredalgae,whicharebothcommerciallyimportant S.J.K.,J.W.K.,U.J.K.,S.L.,G.M.,M.M.,B.J.S.C.O.,H.Q.,J.T.S.,A.G.S.,B.N.S.,V.W.,W.W.,
and have played a major role in the evolution of other algal Z.-Y.W.,C.Y.,S.Z.-R.,Y.Zhuang,Y.Zou,J.W.S.,A.R.G.,andS.E.P.wrotethepaper.
groupsthroughsecondaryendosymbioses. Theauthorsdeclarenoconflictofinterest.
ThisarticleisaPNASDirectSubmission.
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cytoskeleton calcium-signaling carbohydrate-activeenzymes FreelyavailableonlinethroughthePNASopenaccessoption.
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stresstolerance vitaminB12 Datadeposition:TheWholeGenomeShotgunprojecthasbeendepositedatthe
DNADataBankofJapan/EuropeanNucleotideArchive/GenBank(accessionno.
The red algae are one of the founding groups of photosyn- MXAK00000000).Theaccessionno.forthechloroplastgenomeisMF385003.
thetic eukaryotes (Archaeplastida) and among the few mul- 1Towhomcorrespondenceshouldbeaddressed.Email:[email protected].
ticellular lineages within Eukarya. A red algal plastid, acquired 2Presentaddress:GeneticsProgram,UniversityofNewHampshire,Durham,NH03824.
throughsecondaryendosymbiosis,supportscarbonfixation,fatty 3Presentaddress:CollegeofEnvironmentalScienceandEngineering,OceanUniversityof
China,Qingdao266100,China.
acid synthesis, and other metabolic needs in many other algal
Thisarticlecontainssupportinginformationonlineatwww.pnas.org/lookup/suppl/doi:10.
groupsinwaysthatareconsequential.Forexample,diatomsand 1073/pnas.1703088114/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1703088114 PNASEarlyEdition | 1of10
phytes (“seaweeds”) that comprise the majority of described
Significance
species (13). Phylogenomic comparisons of Bangiophyceae
(e.g., P. umbilicalis, P. yezoensis) and Florideophyceae (e.g.,
Fossil evidence shows that red algae (Rhodophyta) are one of C. crispus, Calliarthron tuberculosum) suggest that these two red
themostancientmulticellularlineages.Theirecological,evolu- algal classesarehighlydiverged(SIAppendix,Fig.S12).Theab-
tionary, and commercial importance notwithstanding, few red sence of some pathways and genes from red algae is likely be-
algal nuclear genomes have been sequenced. Our analyses of causeofgenomicreductionintheredalgalancestor(4),andwe
thePorphyraumbilicalisgenomeprovideinsightsintohowthis confirmedthatPorphyralacksgenesdescribedpreviouslyaslost
macrophytethrivesinthestressfulintertidalzoneandintothe in other red algae, including those encoding enzymes of the
basisforitsnutritionalvalueashumanfood.Manyofthenovel glycosyl-phosphatidylinositol(GPI)anchorbiosynthesispathway
traits (e.g., cytoskeletal organization, calcium signaling path- (Kyoto Encyclopedia of Genes and Genomes map00563,
ways)wefindencodedinthePorphyragenomeareextendedto 22genes),autophagyproteins(KOpathwayko04140,17genes),
other redalgal genomes, andour unexpectedfindings offera andmostflagellarproteins(4).
potentialexplanationforwhytheredalgaeareconstrainedto
smallstaturerelativetoothermulticellularlineages. Cytoskeleton.The cytoskeleton of red algae is poorly character-
ized,despitethelong-recognizedabsenceofflagellafromthered
human health is immense. Moreover, the oldest taxonomically algae(14,15).Nuclear-associatedorganellesthatlackcentrioles
resolved multicellular eukaryote in the fossil record (1.2 Ga) is appeartoorganizethemitoticspindle(1),andfreeze-substitution
thebangiophyteredalgaBangiomorpha,whichcloselyresembles reveals cytoplasmic microtubules and bundles of actin microfila-
the extantmarine algaBangia (6).Asis typical of mostbangio- ments (16). Cytoskeletal inhibitors and fluorescent probes (e.g.,
phytes,PorphyragrowsinoneofEarth’smostphysicallystressful FITC-phalloidin for microfilaments) demonstrate that actin mi-
habitats, the intertidal zone, where organisms are exposed to crofilaments form cortical rings during cytokinesis and during
daily and seasonally fluctuating temperatures, high levels of ir- sperm/egg fusion, ensheath migrating secretory vesicles and or-
radiance (including UV), and severe osmotic stress and desic- ganelles, and are prominently labeled in amoeboid red algal
cation.Porphyraanditsancestorshavecompetedsuccessfullyin spores(17–21).Certainly the composition ofthe red algal cyto-
this dynamic and severe environment for over a billion years, skeletonmustdeterminemanyofthecapabilitiesandlimitations
throughnumerouschangesinclimateandmassextinctions. of red algae because of the fundamental roles the cytoskeleton
Here we describe the genome of Porphyra umbilicalis. Exam- plays in intracellular transport, secretion of cell wall materials,
inationofthePorphyragenomeandcompletegenomesofother regulationofcellsizeandshape,andresponsestodevelopmental
red algae [Chondrus crispus (7), Cyanidioschyzon merolae (8), and environmental signals that influence cell polarity and com-
Galdieria sulphuraria (9), Porphyridium purpureum (10), Pyropia plex tissue development in many eukaryotes (22, 23). Here we
yezoensis (11)] revealed numerous additional differences be- report that Porphyra and other red algae have significantly re-
tween the red algae and other eukaryotic lineages, including a duced cytoskeletons and consider the consequences for size
reduced complement of motor proteins, unique signaling mole- andcomplexity.
cules, and augmented stress tolerance mechanisms, especially We identified four closely related actin genes in Porphyra, as
inPorphyra. well as the chromatin remodeling, actin-related protein Arp4,
butnootherArpproteins(SIAppendix,TableS15).Thelackof
ResultsandDiscussion the dynactin complex Arp1 is consistent with loss of a dynein
Genomic Analysis. An 87.7-Mbp assembly of the P. umbilicalis motor, but the general absence of Arp2/Arp3 from red algae is
(hereafter, Porphyra) nuclear genome was generated from surprising. In eukaryotes, Arp2/3 nucleates the formation of
PacBio whole-genome shotgun sequencing, with insertions and branched microfilaments that mediate amoeboid motion (22),
deletionscorrectedusingIlluminawhole-genomeshotgunreads which is observed in many types of red algal spores, including
(SI Appendix, Methods). The Porphyra genome has a substan- Porphyraneutralspores(24)andPyropiapulchellaarcheospores
tialrepeat component (43.9%)for acompact genome, withthe (21). How these spores move without Arp2/3 is an intriguing
most common repeat classes being DNA (15.5 Mbp) and LTR question;perhapstheyrapidlypolymerizemicrofilamentswiththe
(14.9Mbp)elements(SIAppendix,TableS5).Genemodelswere aidofothernucleatingmachinery,suchasformins(22),whichare
predictedat13,125lociusingdenovogenepredictionalgorithms present. However, although formins in many animals, fungi, and
supported by evidence from protein homology and expression plants are members of expanded gene families that have been
data (SI Appendix, Table S6). A typical gene has ∼two exons, differentiated to support processes required to build complex
implyingabundantsplicingforaredalga;however,only235al- morphologies(e.g.,polarizedtipgrowthandcellplateorientation)
ternative splice-forms were identified from expressed sequence (25,26),Porphyrahasonlytwoformins(SIAppendix,TableS15).
tag coverage of genes (SI Appendix, Table S6). Overall, the ge- Inadditiontoformins,Porphyraandotherredalgae(SIAppendix,
nomeis65.8%G+C,butprotein-codingregionsaverage72.9% TablesS15andS16)containprofilin,whichinteractswithformins;
andreachupto94%G+C(SIAppendix,Fig.S7).Nearly98%of cofilin, a key depolymerizing factor; and severin, which cuts mi-
the sequenced transcripts (expressed sequence tags) can be crofilaments to promote remodeling. However, we did not find
mapped to the genome assembly, and we identified complete other well-conserved, widely distributed actin-modifying proteins
complements of genes encoding RNA polymerase subunits and (e.g., WASP/WAVE, CapZ, fimbrin) inPorphyra, and few ifany
all other conserved proteins involved in transcription, trans- convincinghomologsinotherredalgae(SIAppendix,TableS16).
lation, and DNA synthesis (SI Appendix, Table S10), suggesting Themoststriking limitationtomicrofilament-mediatedphenom-
thatthegenome isnearlycomplete. enainPorphyraandmostotherredalgaeistheabsenceofmyosin.
Phylogenomic analysis (12) of the red algae (Rhodophyta) Myosingeneswerenotdetectedinanyoftheavailablegenomesof
distinguishesaclass (Cyanidiophyceae) ofextremophilic unicel- theBFclade(Fig.1),andonlynonspecific(27)myosininhibitors
lular species and two sister clades of mesophilic species, which (2,3-Butanedione monoxime) were used previously (e.g., ref. 21)
we refer to here as the SCRP (Stylonematophyceae, Comp- toinfermyosinactivity.Wedofindthatthesinglemyosinanno-
sopogonophyceae, Rhodellophyceae, Porphyridiophyceae) and tatedpreviously(9)intheextremophileGaldieria,butabsentfrom
the BF (Bangiophyceae, Florideophyceae) (SI Appendix, Fig. Cyanidioschyzon (8), is also found in three classes of the SCRP
S11).TheSCRPcladecontainsunicells,microscopicfilaments, clade,butnotinPorphyridiophyceae(Fig.1andSIAppendix,
and microscopic blades, whereas the BF clade holds macro- Fig. S17).
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Fig.1. Distributionofcytoskeletalmotorproteins(kinesin,cytoplasmicdynein,andmyosin)inredalgaecomparedwithotherselectgroupsofEukarya,
showingthereducedcytoskeletalcapacityofredalgalcells.Standardmotorfamilynomenclaturesfordyneins(104),kinesins(105),andmyosins(106)are
used;forexample,kinesinsK1,K2,andK3arecytoskeletalmotorsthatmoveorganellesandvesiclesinmanyeukaryotes.Footnotes:*Thismaybeacon- N
taminatingsequence.†TheSaccharomycesdyneincomplexhaslostmuchofthefunctionalityfoundinotherorganisms(104).‡Additional,flagellar-related TIO
dyneinHCarenotshownandareabsentinredalgaeandmanyplants.§MyosinsM8andM11arevesicletransportersinplants.¶GaldieriaandSCRmembers LU
O
oftheSCRPclade(SIAppendix,Fig.S11)haveamyosinsimilartomyosin27ofapicomplexans. EV
We found that the Porphyra genome encodes the expected Fig.1)canactasaminus-enddirectedtransporterinlandplants
α- and β-tubulin proteins, as well as some proteins related to (reviewed by refs. 29 and 30). In addition, a few red algae do
tubulin folding (e.g., Porphyra contains cofactors B and D, but contain members of the kinesin 4 subfamily (Fig. 1), which are
notAorE)andmicrotubulenucleation(e.g.,γ-tubulin,γ-complex reported to be plus-end directed microtubule vesicle motors in
proteins)(SIAppendix,TablesS15andS16).However,manyof plants (31). However, Porphyra and most other sequenced red
the expected tubulin regulatory proteins that are widely distrib- algae lack this protein. Thus, unless kinesin 5 or kinesin 7 has
uted across eukaryotes and even found in other red algae unexpectedfunctionality,Porphyraandthemajorityofredalgae
are missing. For example, Porphyra contains EB1 and Mor1/ (BFclade)wouldalsoapparentlylackaplus-enddirectedkinesin-
XMAP215, two highly conserved proteins of the MT plus-end microtubule motorsystem. Taken together,thesedata show that
tracking (+TIP) complex, but the +TIP CLASP and the cross- thepaucityofmotorsexplainstheabsenceofcellstreamingfrom
linker MAP65 appear absent, even though these highly con- redalgaeincludingPorphyra.
servedgenesarepresentinotherredalgae(SIAppendix,Table Onecounterpoint tothe frequent absenceofnear-ubiquitous
S16). Unknown as yet is whether Porphyra simply lacks these cytoskeletal proteins is that Porphyra does have two septins (SI
activities, or has recruited other proteins to fill their roles. Por- Appendix, Tables S15 and S16), filament-forming proteins that
phyra and other red algae lost flagellar and cytoplasmic dynein areinvolved incytokinesis,cell polarity, andmembrane remod-
motors, intermediate chains, and most light chains. A dynein eling (22, 23). Regardless, our overall analysis of Porphyra and
heavy chain reported from C. crispus might be from a contami- otherredalgaewithsequencednucleargenomes[Chondrus(7),
nant or horizontal gene transfer (SI Appendix, Table S16). Por- Cyanidioschyzon (8), Galdieria (9), Porphyridium (10), Pyropia
phyra and other red algae retain a particular light chain that is (11)] indicates that the red algal cytoskeleton lacks the com-
also conserved in flowering plants, which independently lost plexity and diversity of cytoskeletal elements present in other
flagellar motility (SI Appendix, Table S16). This dynein light multicellular lineages (Fig. 1). Although it is possible that reg-
chain is expressed under abiotic stress and phytohormone ulatory proteins are simply too divergent to recognize, the pau-
treatments in plants (28); thus, it may have a role in the stress city of motors is especially apparent. We suggest that this
toleranceofPorphyra.Incontrasttothelossofthedyneinmotor, observationcouldhelptoexplainlong-standingquestionsabout
Porphyra does have representatives of several kinesin motor morphological evolution in the red algae, including the lack of
subfamilies,specificallykinesins5,7,and14,whichareexpected parenchyma inthese organisms. Compared withother multicel-
to be involved in spindle assembly, kinetochore function, and lular lineages (green algae/plants, brown algae, fungi, animals),
regulation of microtubule dynamics, respectively (Fig. 1 and SI theabilitiestoformlargecellsandlargemulticellularstructures
Appendix, Fig. S18 and Tables S15 and S16). Porphyra also appear to be limited in the red algae. For example, the largest
contains three divergent kinesins that usually group with the cellsinPorphyraarefoundinitsholdfast,whichiscomposedof
mitotic motor kinesin 13. However, classic vesicle transport thousands of thread-like, slow-growing rhizoid cells that are
motors (kinesin subfamilies 1, 2, and 3) appear to be absent in millimeterslong,andsomespeciesofGriffithsia(32),whichisa
Porphyra,which is particularly surprising considering the appar- subtidalflorideophyte,havecells∼2-mmlong.Incontrast,large
entlossofmyosins anddyneins. cells filling special niches or functions evolved in multiple
Giventhesurprisingpaucityofmotors,howdoPorphyracells freshwaterandmarinegreenalgae,includingcoenocytes(1),as
accomplishintracellulartransportofmembranesorothercargo? germinatingpollentubesinlandplants(33),assporangiophores
One answer may be provided by the observation that the “mi- infungi(34),asnervecellsinanimals(35),andassieveelements
totic” motor kinesin 14 (present in all sequenced red algae, see inbrownalgae(36).Maintenanceoflargecellswouldbeexpected
Brawleyetal. PNASEarlyEdition | 3of10
torequirevigorousmultidirectionalintracellulartransport,which Porphyraencodesgenesforcatalasesandperoxidases,aswell
seemsunlikelywith amotorrepertoireaslimited as that seen in as the biosynthesis of numerous antioxidants, such as ascorbic
Porphyraandtheotherredalgaewithsequencedgenomes(Fig.1). acid (vitamin C) and tocopherol (vitamin E) (SI Appendix, Ta-
Similarly, brown algae (46-m kelps) (36), animals, and plants bles S20–S22). When overexcitation of photosynthetic electron
assemble large, complex 3D body plans with true parenchyma, transport occurs and reactive oxygen is generated, catalase de-
but multicellular forms of red algae mostly consist of simple toxifieshydrogenperoxideinredalgae,andtheexpansionofthe
filaments or filaments interwoven and tacked together by sec- catalase gene family in Porphyra and Pyropia (five genes) (SI
ondary pit plugs (1) (i.e., pseudoparenchyma). Red algae are Appendix,TableS21)comparedwithotherredalgae(onetotwo
usually≤50-cmlongandonlyafewspeciesreach2minlength genes) could reflect the demand for detoxification of reactive
(36, 37). Fungi cannot make parenchyma, but saprophytic my- oxygenspeciesthatcannotdiffuseawayfrombladesexposedby
celiagrowto≥50-mlength(38).Failureoftheredalgaetoform thefallingtidetohighlightandairwhiletheyarestillhydrated
large multicellular structures is not straightforwardly attributed andphotosyntheticallyactive.Tocopherolspreventphotooxidative
to cytoskeletal limitations; however, plants, which retain two damage of polyunsaturated fatty acids (45), and the Porphyra
motors (myosin, kinesin) despite their independent loss of dy- γ-tocopherol methyl transferase (SI Appendix, Table S22) cata-
nein, suffer serious stunting and other developmental abnor- lyzessynthesisofα-andβ-tocopherol.Theisomercompositionis
malities following gene knockouts of myosins (23, 39). Analysis unknown,butα-tocopherolhasbeenidentifiedinPorphyrablades
of major transcriptional and developmental regulators does not and is considered the more potent antioxidant (46, 47). The
offer a compelling explanation for why red algae have failed to 32 heat shock proteins (Hsp) in Porphyra indicate a possible ex-
evolve tissues comparable to those in brown algae, plants, and pansion of the Hsp40 family (SI Appendix, Table S20), which
animals (40). Taken together, our comparative analysis of the are cochaperones of Hsp70 and play an important role in
genomes of Porphyra and other red algae leads us to speculate protein maturation and repair under normal and stressed
that the small number of cytoskeletal elements in red algae conditions (48, 49).
comparedwiththoseinothermulticellularlineages(Fig.1)has Porphyra is frequently exposed to elevated intensities of
constrained the ability of red algae to develop larger, more UV radiation in the intertidal zone and shows remarkable
complexcellsandmulticellularstructures. tolerance to both UV-A and UV-B (50, 51). Porphyra has at
least two strategies to protect photosynthesis and other key
Stress.TheecologicalsuccessofPorphyraandmanyoftheclosely cellular processes from UV damage: mycosporine-like amino
related bangiophyceans (37, 41) in the intertidal zone suggests acids (MAAs) and circadian control over the timing of UV-
that these species developed cellular mechanisms to cope with sensitive processes.
this harsh environment. In particular, Porphyra grows from the MAAsactas“sunscreens”andcompriseupto1%ofthedry
mid-to-highintertidalzone,whereitisroutinelyexposedduring weight of Porphyra, with the compound porphyra-334 being the
dailylowtidestohighlight,desiccation,andextremefluctuations major MAA (51). Four proteins—MysA, MysB, MysC, and
in temperature and salinity. Blades can lose up to 95% of their MysD—support the biosynthesis of the MAA shinorine in cya-
wateronsomedays,butaremetabolicallyactiveassoonasthey nobacteria,suchasNostoc(52,53),whereasMysDisreplacedby
are rehydrated by the rising tide (24). Here we infer novel a nonribosomal peptide synthase in Anabaena (Fig. 2). Cyano-
adaptations to cope with these stresses. bacterial MysD shows a relaxed substrate specificity, with con-
densation of threonine instead of serine onto mycosporine-
Photoprotection.Light is required for photosynthesis, but severe glycine to yield porphyra-334 (53). The Porphyra genome
cellular damage can result from exposure of photosynthetic or- containsageneencodingaMysAandMysBproteinfusionthat
ganisms to the high levels of light (visible and UV) that are isalsofoundinseveralotherredalgaethatsynthesizeMAAs,as
present in the mid-to-high intertidal zone where most bangio- well as in some dinoflagellates, which were proposed to have
phyceanalgae,includingPorphyra,grow.Porphyra hasthesame acquired the still separate but neighboring genes from a cyano-
complement of genes (SI Appendix, Table S19) to carry out bacterium(54)(Fig.2andSIAppendix,Figs.S23andS24).The
photosynthesis as other red algae, but exhibits a notable set of presence of the MysA–MysB fusion in red algae suggests that
photoprotection strategies. Among the 13 genes encoding chlo- dinoflagellates could have acquired these genes from red algae
rophyll a-binding light-harvesting complex proteins are two through secondary or serial endosymbiosis (54) (SI Appendix,
“RedCap” genes (42), which may be involved in reorganization Fig.S23).Moreover,Porphyraandrelatedspeciescontainagene
ofthephotosynthetic antennaduringtheshiftfromdarknessto encoding a MysC–MysD fusion protein, and in the Porphyra
light (43). Porphyra also has 11 genes encoding “high light-in- genometheMysA–MysBandMysC–MysDfusiongenesarenext
duced”or“one-helix”proteins(hereOHPs)thathaveessential to each other but transcribed on opposite DNA strands with
roles for photoacclimation and cell viability under stressful en- adjacent 5′-ends (Fig. 2).Although theChondrus genomealso
vironmental conditions (43, 44). Mechanistically, OHPs may contains clustered MysA–MysB and MysC–MysD, the fusion
regulate chlorophyll and tetrapyrrole biosynthesis, stabilize proteins are encoded on opposite strands with the 3′-ends of
photosystem I (PSI), bind free chlorophyll or chlorophyll- the genes adjacent. Conservation of the MAA gene cluster in
breakdown products from damaged PSII complexes during the PorphyraandChondrusandthetwogene-fusioneventssuggest
damage/repair cycle, or bind carotenoids that dissipate excess that this arrangement provides a selective advantage and effi-
absorbed light energy. In contrast to the 11 Porphyra OHPs (SI cient MAA biosynthesis for red algae that experience high
Appendix, Table S19), we found 4 OHPs in Chondrus, which UV irradiance.
experiences less drying and light stress because of its low in- Developmental and abiotic stress responses are often associ-
tertidal/subtidalhabitat,6inP.yezoensis,7inPorphyridium,and ated with photoreceptors in eukaryotes. The plant circadian
only1OHPinCyanidioschyzon,whichinhabitsastablehotspring clock contains blue- and red-light photoreceptors, including
environment(SIAppendix,Photosynthesis,Photoprotection,Stress cryptochromes(CRY)andphytochromes(PHY),toentrainthe
Genes). More analysis is needed, but the putative gene family circadian clock (55, 56); these photoreceptors are also involved
expansion in Porphyra suggests positive selection for increased in other fundamental processes in plants, including growth and
gene dosage. Porphyra was one of the first organisms where development. Porphyra does not appear to encode a PHY pho-
quenchingofexcessexcitationenergyinresponsetodesiccation toreceptororatypicalplantCRYphotoreceptor,althoughithas
stress was observed (44), but the molecular mechanisms re- maintained four genes of the CRY/photolyase family. The Por-
sponsibleforthisquenchinginredalgaeremain unclear. phyra CRY that is most like plant CRYs is similar to a DNA
4of10 | www.pnas.org/cgi/doi/10.1073/pnas.1703088114 Brawleyetal.
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Fig. 2. Conserved gene clusters involved in MAA biosynthesis in P. umbilicalis and related red algae. (A) Biosynthetic pathway from sedoheptulose OL
7-phosphatetoshinorineincyanobacteriaandproposedpathwaytoporphyra-334inredalgae.(B)Comparisonofgeneclustersandgenefusionsinthe EV
cyanobacteriaAnabaenaandNostocandtheredalgaeP.umbilicalisandC.crispus.
photolyase(PHR2)(SIAppendix,Fig.27andTableS26)(57).In amino acid derivative (SI Appendix, Table S31). Porphyra also
contrast,otherredalgaeseemtoencodeplantCRYs;however, has a wide range of ion transporters, including an unusual class of
thesegroupcloselytothecyclobutanepyrimidinedimerclassIII Na+/H+ exchangers that are most similar to the NhaP class of
photolyases(58)insomecases(SIAppendix,Fig.S27).Porphyra transportersfromα-ProteobacteriaandaredistinctfromtheNa+/H+
alsoencodesaclassIIcyclobutanepyrimidinedimerphotolyase exchangerspreviouslyidentifiedineukaryotes.Porphyrahastwo
(Pum0022s0035.1) as well as a CRY-DASH protein (59) P2C-type Na+/K+ ATPases and a P3A H+-ATPase similar to
(Pum2644s0001.1)inaseparatesubfamilyofCRYsthatdisplays those in land plants, suggesting Porphyra is able to energize its
DNArepairactivity;theseproteinsarepresentfrombacteriato plasma membrane with either Na+ or H+ for secondary active
vertebrates (59). In addition, there is an animal-like CRY transport, which could aid its survival in the intertidal zone (SI
(Pum0401s0002.1) encoded on the Porphyra genome (SI Ap- Appendix, Table S31). The Porphyra genome also contains a
pendix,Fig.S27).Animal-likeCRYscouldfunctionasblue-light range of Ca2+-permeable membrane channels that could play a
photoreceptorsassociatedwiththeentrainmentofthecircadian role in osmotic stress signaling, including two homologs of
clock, as they do inDrosophila and someotherinsects,orcould OSCA (SI Appendix, Table S31), a recently identified Ca2+
even add an oscillatory component to the clock, as in mouse or channel in land plants (68); however, the Ca2+ sensor kinases
humans(59).Theanimal-likeCRYinPorphyraiscloselyaffiliated through which Porphyra senses and responds to cytosolic Ca2+
withthecryptochromephotolyasefamily(e.g.,Ostreococcustauri,
elevations appear to be distinct from land plants and, indeed,
Phaeodactylum tricornutum in SI Appendix, Fig. S27), which has
fromthose ofallothereukaryotes.
maintainedphotolyaseactivity(60,61),incontrasttootherplant Land plants possess two expanded families of Ca2+ sensor
and animal-like CRYs. Moreover, cryptochrome photolyase kinases, the Ca2+-dependent protein kinases (CDPKs) and the
family CRY can affect transcriptional activity in a heterologous
calcineurin B-like protein (CBL)-interacting protein kinases
clock system (e.g., mammalian CRY) and control blue-light–
(CIPKs), which are activated through the binding of CBL (69,
dependent cellular processes, as found with insect or plant CRYs
70).TheCIPKshavebeencharacterizedextensivelyinthegreen
(60,61).SimilartotheChlamydomonasreinhardtiianimal-likeCRY
lineage(Viridiplantae),andtheyarepresentinothereukaryotes,
(aCRY), the Pum0401s0002.1 protein could sense blue light and
including stramenopiles, haptophytes, and excavates, suggesting
possiblyotherlightqualities,includingredlight(62,63);thisred-
alikelyoriginearlyineukaryoticevolution(71).Surprisingly,we
light sensing ability stems from the formation of the neutral
radicalformofthephotoreceptor’sflavin chromophore. foundthatgenesencodingCIPKsandCBLsareabsentfromthe
Porphyragenomeandfromallotheravailableredalgalgenomes
Signaling and Homeostasis. Porphyra must cope with significant and transcriptomes. The CDPKs are also absent from Porphyra
osmotic and ionic stress in the intertidal zone. Several low mo- and notably from other bangiophytes and florideophytes, but
lecular weight carbohydrates act as compatible solutes in red theyarepresentinredalgaeintheSCRPclade(Fig.3;compare
algae, including Porphyra (64–66). We found enzymes encoded SI Appendix, Fig. S11 and Table S32). Another class of Ca2+
on the Porphyra genome that support floridoside and iso- sensor kinases, the Ca2+/calmodulin-dependent protein kinases
floridoside synthesis, but no evidence for digeneaside synthesis (CAMKs), which are important in both plants and animals, are
(SIAppendix,TableS31).TaurineisalikelyosmolyteinPorphyra also missing in Porphyra. This finding suggests that much of
(67), and we found homologs of enzymes, such as cysteine the extensive network of Ca2+ sensor kinases found in other
dioxygenase,implicatedinmetazoantaurinebiosynthesis,which eukaryotes is absent in the red algae (Fig. 3). Whereas the
suggests an ancient origin for eukaryotic biosynthesis of this Porphyragenomeencodesseveralproteinswithdomainssimilar
Brawleyetal. PNASEarlyEdition | 5of10
PP2C in the abscisic acid (ABA) signaling pathway (75, 76).
Animals CaMK Furthermore, there are homologs of several ABA biosynthetic
K
M genes in Porphyra (SI Appendix, Fig. S35A), and ABA synthesis
A Plants CCaMK and responses to exogenous ABA are reported for Pyropia
C
orbicularis(77).Althoughhighlyspeculative,theevidenceraises
Porphyra CaMK-like
the possibility that the SnRK family plays important roles in
stressresponsesincludingABA-mediatedresponsesinPorphyra.
Genomic data also support the presence of ethylene-mediated
Plants CDPK regulation in Porphyra, because the Porphyra genome contains
K genesencodingseveralproteinsinvolvedinethylenebiosynthesis
PD Porphyra CDPK-like (SIAppendix,Fig.S35B),andethylene wasdetectedandshown
C
toinducestressresponsesinredalgae(78).
Porphyridium CDPK
The cell cycle is regulated by dimers of cyclins and cyclin-
dependent kinases (CDKs). Except in C. merolae, where circa-
dian rhythms and stress responses regulate the G /S transition
1
Plants CIPK (79), the red algal cell cycle is not well characterized. Results
K
P here, coupled with the earlier studies of C. merolae, show that
IC Porphyra CIPK-like regulation of the red algal cell cycle is similar to that of meta-
zoans and plants (80, 81), except that we did not find cyclin D
encodedinanyredalga,includingPorphyra(SIAppendix,Table
KL Porphyra CDTKL S37).CyclinDsarewellconservedinallothereukaryoteswhere
T they regulate the G1/S transition. The mitotic-specific cyclin A
CD Chondrus CDTKL (CYCA),aknowncell-cycleprogressionregulator,mightinstead
function in place of D-type cyclins in red algae. We found that
the glaucophyte (Archaeplastida) Cyanophora paradoxa (2) also
lacksacyclinDhomolog.
Serine/threonine kinase NAF domain
CaM binding KA1 domain PlantDefenseGenes.Bangiophyte pathogens include some oomy-
cetes,viruses,andbacteria,andtheseorganismssometimescause
CAMK AD domain TKL protein kinase
serious economic losses to nori aquaculture. Land plants detect
EF-hand Ankyrin
pathogens via an array of cell surface pattern-recognition recep-
Pleckstrin-homology tors (e.g., receptor-like kinases) and intracellular receptors (e.g.,
nucleotide-bindingdomainandleucine-richrepeatproteins)(82).
Fig.3. ModulestructureandpresenceorabsenceofdifferentCa2+sensor
We found no evidence for this higher plant type of pathogen
kinasesinPorphyraandothereukaryotes:CAMK,CIPK,CDPK,CDTKL.The
detection,buttwofamiliesofintracellularligand-bindingprotein
CDTKLsfromPorphyraandotherredalgaerepresentanewlyrecognized
familyofproteinkinasesbelongingtotheTKLfamilythatcontainsmultiple containingNB-ARCdomains,andafamilyofpotentialextracel-
Ca2+-bindingEFhands. lularreceptorscontainingmalectinandIg-likefold-domains,were
found in the multicellular red algal genomes (SI Appendix, Fig.
S38).Wealsofoundabangiophyte-specificfamilyofatleastthree
tothekinase domains ofCAMKs,CDPKs,andCIPKs, thereis proteinsharboringvWFAandC-typelectindomains(SIAppendix,
no indication that these proteins are regulated by Ca2+ or Table S39); C-type lectin domains are involved in pathogen de-
Ca2+-bindingproteins.Instead,wefoundthatPorphyrapossessesa tectioninsomeanimals(83,84).
class of Ca2+ sensor kinases with two to three Ca2+-binding EF-
hand domains at the N terminus and a kinase domain at the C Cell Walls. Red algae synthesize many unique polysaccharides,
terminus(Fig.3andSIAppendix,TablesS32andS33).Thekinase suchasagarsthatcontributetotheirecological successandare
domainfromallknownCa2+sensorkinasesbelongstotheCAMK of significant interest to biotechnological and industrial appli-
group of kinases, whereas this uncharacterized protein belongs to cations. The cells ofthe blade of Porphyra spp. formanonrigid
thetyrosinekinase-like(TKL)classofkinases(72,73).Thegenes cell wall that plays an important role in osmotic acclimation by
encodinghomologousproteinsarepresentonmanyotherredalgal allowing dramatic changes in cell volume without plasmolysis
genomes, including Chondrus, Cyanidioschyzon, and Galdieria, but (85,86).Theskeletalcomponentofthecellwallofbladecellsis
appear to be absent from all other characterized eukaryotic ge- notcellulosebutpartiallycrystallineβ-1,4-linkedmannaninthe
nomes. Hence, the Ca2+-dependent TKLs (CDTKLs) represent a outercellwall,withahighlycrystallineinnerlayerofβ-1,3-linked
newlyrecognizedclassofCa2+-regulatedkinasesthatappeartobe xylan (87, 88). We discovered two glycosyltransferase (GT2)
uniquetoredalgae(Fig.3). enzymes(Fig.4andSIAppendix,TableS40)inPorphyra thatare
Thesucrosenonfermenting-1–relatedkinase(SnRK)familyof closely related to plant cellulose synthase-like (CSL) CSLAs
serine/threonine kinases plays important roles at the interface (mannan synthases) and CSLCs (xyloglucan synthases) and the
betweenmetabolicandstresssignalingfromfungi(yeast)toland greenalgalCSLsproposedtobeimplicatedinmannansynthesis
plants (74). There are 38 SnRKs divided into three subclasses (89, 90). These Porphyra GT2 enzymes represent excellent can-
(SnRK1,-2,-3)inArabidopsis,andthePorphyragenomeencodes didates for mannan synthases, and their discovery in Porphyra
31proteinswithhomologytothekinasedomainsofArabidopsis suggeststhatbothCSLAandmannanbiosynthesisoriginatedin
SnRK proteins (SI Appendix, Fig. S34). Phylogenetic analysis thelast-commonancestorofgreenandredalgae,ratherthanin
places the Porphyra SnRK group in two clades that include the Viridiplantae, as previously proposed (89). These findings
Arabidopsis SnRK1 and SnRK3/CIPK, and a third expanded suggest that mannans had an ancestral function in red algae,
clade containing 19 unique members (SI Appendix, Fig. S34). althoughitappearstheFlorideophyceaeabandonedmannansfor
Although no Porphyra SnRKs are most similar to the SnRK2 cellulose,whereastheBangiophyceaealternatebetweenamannan/
subfamily of Arabidopsis, several members of the SnRK3/CIPK xylan-basedcellwallinthegametophytesandacellulose-basedcell
clade contain motifs similar to the C-terminal domain II of wallinthefilamentoussporophytes.ThePorphyragenomehastwo
SnRK2(SIAppendix,Fig.S34),whichmediatesinteractionwith genes encoding glycosyl hydrolases (GH) of the GH113 family,
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80 CESA (Bryophytes, Lycophytes, Angiosperms)
99
CSLD (Bryophytes, Lycophytes, Angiosperms)
100
CSLB (Eudicots)
100
95 CSLH (Monocots)
100100CSLG (Eudicots)
81 CSLE (Angiosperms)
100 100
CESA (Oomycetes)
94 CHC T00008424001 Chondrus crispus
93 CESA Griffithsia monilis
CHC T00009301001 Chondrus crispus
100 Pum0564s0005.1 CESA (red algae)
Pyropia yezoensis CesA
100
82 Pum0678s0001.1
98 Porphyra purpurea esContig8639
CESA (Amoebozoa)
100 100
CESA (brown algae and eustigmatophyceae)
100 contig 8821 g2086 Pyropia yezoensis
85 Pum0231s0012.1
82 100 contig 3060 g650 Pyropia yezoensis CSL (Bangiophyceae)
Pum0372s0002.1
100 100 CSL (Chlorophyta green algae)
100
100 CSLA (Bryophytes, Lycophytes, Angiosperms)
85 CSLC (Bryophytes, Lycophytes, Angiosperms)
100
100 bcsA (Cyanobacteria) ON
contig 34751 g8353 Pyropia yezoensis CSL (Bangiophyceae) UTI
100 Pum0053s0020.1 L
O
65 bcsA (bacteria) EV
0.5
Fig.4. Neighbor-joiningphylogenetictreeofGT2showingthepositionsofPorphyrasequences(darktriangles)androotedonbacterialcellulosesynthase
(bcsA).Redalgalcellulosesynthases(CESA)clustermostcloselywithstramenopileoomyceteCESAs,whereasbangiophyteCSLenzymesaresistertoacladeof
greenplantCSLsthatincludesamannansynthase.TworedalgalGT2sarerelatedtobcsA,likelybecauseofhorizontalgenetransfer.Redalgalsequenceslie
onredbranchesandtrianglesgroupsequencesinViridiplantae(green),stramenopiles(brown),Cyanobacteria(turquoise),Bacteria(pink),andAmoebozoa
(gray).TheproteinswerealignedusingMAFFTwiththeL-INS-IalgorithmandthescoringmatrixBlosum62andthenmanuallyrefinedwithBIOEDIT.The
phylogenetictreewascalculatedusingmaximumlikelihoodinMEGA6.06.SeeSIAppendix,TableS42forfullnamesandGenBankaccessionnumbers.
Bootstrapvalues≥65%areshownatnodes.
which are predicted to be β-mannanases, indicating that Porphyra nismofconvergentevolution(94).Thisspeakstothecriticalrole
hastheenzymesrequiredforboththebiosynthesisanddegradation ofsulfatedECMsinhigh-salinityenvironments(physiologicalsa-
ofmannanspresentinthecellwallsoftheblade. line and seawater). The Porphyra genome encodes three GH16
Theextracellularmatrix(ECM)componentofthecellwallin enzymes that form a clade with other red algae and with the
Porphyra is a highly hydrated agar called porphyran that limits agarases and porphyranases from marine bacteria (SI Appendix,
desiccation and contributes to the flexibility of the cell wall Fig. S41). These enzymes likely function in cell wall remodeling
during osmotic stress (91) (SI Appendix, Figs. S41 and S45 and and are unknown in unicellular red algae, which supports the
Table S40). Porphyran is mainly composed of porphyranobiose possibleinvolvementofbacteria intheevolution ofmulticellular
but also contains varying percentages of agarobiose (92). The algae(95).
Porphyragenomeencodesafamily2carbohydratesulfotransferase
(ST2) that may act as a L-galactose-6-O-sulfotransferase in ObtainingNutrientsatHighTide.WefoundthatPorphyrahasgenes
the biosynthesis of porphyran (SI Appendix, Table S40). After forFe-uptakemechanismsthatlikelylendspecificityandenable
C. crispus, the closest related homologs to this red algal sulfo- high-affinity assimilation during the narrow tidal window when
transferase are found in metazoans (Nematostella vectensis, sea blades are underwater (Fig. 5 and SI Appendix, Table S46). In
anemones)andtheclosestcharacterizedenzymesaredermatan addition to several putative Fe2+ transporters of the NRAMP
and chondroitin 4-O-sulfotransferases; similarly, the Porphyra and ZIP family proteins (which are typically broad-specificity
GT7,aftertheC.crispusGT7,ismostsimilartometazoanGT7s divalent metal transporters) (96), the Porphyra genome en-
(Acropora digitifera, staghorn corals) and the closest character- codes a high-affinity iron transport complex containing a per-
ized enzyme is chondroitin sulfate N-acetylgalactosaminyl- mease(FTR1)andmulticopperoxidase(FET3inyeast/FOX1in
transferase2(Homosapiens).Thesefindingslendsupporttothe green algae) and members of the FEA and ISIP2a families.
ancestralnatureofthebiosynthesisofsulfatedpolysaccharidesin The latter are related algal-specific protein families containing
the eukaryotes. Homologous carbohydrate sulfotransferases are secreted soluble proteins involved in iron assimilation and
indeed conserved at least in metazoans, brown algae, and red membrane-bound iron-uptake facilitators (97, 98). Although
algae,soweremostlikelypresentinthelast-commoneukaryotic both iron-uptake strategies are found in microalgae, we were
ancestor(91,93).Genesencodingcarbohydratesulfotransferases surprised to find FTR1 and FOX1 homologs in a macroalga,
are not found in sequenced genomes of terrestrial plants and given the absence of this complex in higher plants and animals.
freshwateralgae,butmarineangiospermshave,relativelyrecently, Basedonananalysisofphylogeneticdistribution,FTR1homologs
readapted to the marine environment by developing an ECM are found in each of the three main Archaeplastida lineages
containing sulfated polysaccharides through an unknown mecha- (Viridiplantae,Glaucophyta,andRhodophyta)butwerelostafter
Brawleyetal. PNASEarlyEdition | 7of10
traits: genes: cobalamin,theparticularchemicalvariantofB12thatisbioactive
legend:tgreanit ea basbesnetnt tgreanit ep rperseesnetnt algal multicellularlack true roots FTR1 FEA/ISIP2a fSsatohun6repd0p)efs.rauuecIkrsntvasiervtCydhaohaluttlwinacamPdsaeoylrrddgpeoeahpmneye,vroaniarndonaiensdsnm,ftirMneuendpqEteouaTeneldElnytthhlreyeeuxrlmpehmrveaeaaanslntlss-ytiso(tslnrSeteavIsbewsAlleaespdsoMpffeioEnnhudTeiniatHxdst,s(uTtt1opra0epb2bsel)seer;,
Glycine max
intertidal habitat might similarly explain why it retains both
Arabidopsis thaliana
isoforms of this enzyme. Interestingly, the nutritional value of
s Vitis vinifera
nt Embryophyta Zea mays Porphyra appears to be directly related to the nutrient require-
a
pl Oryza sativa mentsforsurvivalinaharshhabitat.
d Selaginella moellendorffii
n
a Sphagnum fallax Summary
l
Physcomitrella patens The red algae have long commanded attention because of the
Marchantia polymorpha 1.2-Ga age of the multicellular bangiophytes, the unique in-
Charophyta Klebsormidium flaccidum
tricacies of their life histories, and their economic importance,
Volvox carteri
butgenomicanalysisofPorphyrasharpensourunderstandingof
Chlamydomonas reinhardtii
Chlorophyta
Chlorella sp. NC64A just how different the red algae are from other eukaryotes. Be-
Coccomyxa subellipsoidea causethecytoskeletonissocentraltogrowth,development,and
Glaucophyta Cyanophora paradoxa the ability to respond to environmental signals, the paucity of
Porphyra umbilicalis cytoskeletalelementsinPorphyraandotherredalgaeisstriking.
e Pyropia yezoensis Only kinesin motor proteins are universally present; dynein
ga Rhodophyta Chondrus crispus motorsareabsent,andmostoftheredalgae,includingPorphyra,
al Porphyridium purpureum appear to lack myosin. This minimal cytoskeleton offers a po-
Cyanidioschyzon merolae
tential explanation for the extreme reduction in stature and
Galdieria sulphuraria
Phaeodactylum tricornutum complexityofredalgaecomparedwithmostothermulticellular
Thalassiosira pseudonana lineages. The unique calcium-dependent signaling pathway
Stramenopiles
Aureococcus anophagefferens further suggests that Porphyra may use distinctive mechanisms
Ectocarpus siliculosus to sense and respond to its environment. Most bangiophytes
Haptophyta Emiliania huxleyi including Porphyra live under harsh intertidal conditions.
Our discovery of ancestral mechanisms of cell wall formation,
Fig.5. PhylogeneticprofileofFTR1andFEA/ISIP2ahomologsinphotosyn-
an expanded array of UV/high-light/reactive oxygen species/
theticeukaryotes.Whethereachorganismisanalga,ismulticellular,orlacks
truerootsisindicatedwithacoloredsquare,andthepresenceofaFTR1or thermalprotectionstrategies,andawealthofnutrienttransporters
FEA/ISIP2ahomologinthatorganismisindicatedwithacoloredcircle.The encoded by the Porphyra genome helps to explain how these red
samecolorisusedtohighlightthecooccurrencebetweenatraitandagene. algaehavethrivedforoverabillionyearsinthepoundingwaves,
Among sequenced algae and plants, FTR1 homologs are only encoded in bakingsun,anddryingwindsofthehighintertidalzone.
genomesoforganismsthatlacktrueroots,whereasFEA/ISIP2ahomologsare
onlyencodedbyalgalgenomes.Multicellularitydoesnotcorrelatewiththe Methods
presenceofeitherproteinfamily.TherootsofSelaginellamoellendorffiiare
P. umbilicalis was isolated as an unialgal culture from a blade growing at
morphologically and evolutionarily distinct, but share characteristics with
SchoodicPoint,Maine(44°20′1.68′′N;68°3′29.14′′W)onApril3,2008.This
truerootsincludingarootcapandroothairs.
singleisolate(103)wasclonedbycultureofprogenyfromitsasexualneu-
tral spores, followed by DNA extraction, and assembly of the genome
from sequences produced using Illumina and Pac-Bio sequencing plat-
the transition to land when plants evolved true roots (Fig. 5).
forms(seeSIAppendix,Methodsforfulldetails).ThisWholeGenomeShotgun
Other inorganic and organic nutrient uptake and conversion ca-
project has been deposited at DDBJ/ENA/GenBank under accession no.
pabilities appear similar to those described for other red algae
MXAK00000000.TheversiondescribedinthispaperisversionMXAK01000000.
(e.g., Fig. 5 and SI Appendix, Fig. S51 and Tables S49, S52, and
S53), except that the number of ammonium transporters (seven ACKNOWLEDGMENTS.Wearegratefultotheanonymousreviewersand
genes) (SI Appendix, Table S49) and intracellular copper trans- editor for their insightful and constructive suggestions. We thank Dr.
LilibethMiranda,CharlotteC.T.Quigley,andCharlotteRoyer(University
porters [P -type ATPases; six genes (MTA1-6)] (SI Appendix,
1B ofMaine)fortheirmajorassistanceinmaintainingculturesofthegenomic
Fig.S47andTableS46)isgreaterinPorphyra,perhapsreflecting strainduringtheprojectperiod;Drs.JoyceLongcore(UniversityofMaine)
themetabolicdemandsoflifeintheupperintertidalzone. and M. Blackwell (Louisiana State University) for discussions about fungi;
Porphyra(“laver”)andrelatedgenera(i.e.,Pyropia,“nori”)are Dr. Sarah Tepler Drobnitch for discussion about kelp sieve elements; and
the many colleagues who provided useful discussion as participants in the
commercially important human foods based on their high min-
Porphyra Research Coordination Network (supported by NSF RCN 0741907
eral, protein, and vitamin content (99) (see Fig. 5 and SI Ap- [toS.H.B.,A.R.G., andJ.W.S.]),especiallyto Elisabeth Gantt (Universityof
pendix, Figs. S51 and S59 and Tables S55 and S56 for related Maryland),anoriginalco-PIoftheNSFRSNandDOEJGIcontract.Thework
findings).NoeukaryotesynthesizesvitaminB (cobalamin),but conductedbytheUSDepartmentofEnergy(DOE)JointGenomeInstitute,a
12 DOEOfficeofScienceUserFacility,wassupportedbytheOfficeofScienceof
Porphyra is a rich source of this organic micronutrient. Vitamin theUSDOEunderContractDE-AC02-05CH11231(toS.H.B.,E.G.,A.R.G.,and
B is a required cofactor for B -dependent methionine syn- J.W.S.).OthermajorresearchsupportwasprovidedbyNSF0929558(toS.H.B.
12 12
thase(METH),andPorphyraencodesbothMETHandMETE, andA.R.G.);National Oceanic and Atmospheric Administration (NOAA)
ContractNA060AR4170108(toS.H.B.);GermanResearchFoundationGrant
the latter a B -independent isoform of methionine synthase
12 Mi373/12-2ofFOR1261(toM.M.);theFrenchNationalResearchAgencyun-
(100).Methioninesynthaseplaysavitalroleinlinkingthefolate derIDEALGGrantsANR-10-BTBR-04-02and04-04“Investissementsd’avenir,
cycle,essentialforDNAmetabolism, andthe methylationcycle Biotechnologies-Bioressources”(toJ.C.,E.F.-B.,G.M.,andS.M.D.);theNew
HampshireAgriculturalExperimentStation,ScientificContributionNo.2694,
responsible for production of S-adenosylmethionine, the uni-
supportedbytheUSDepartmentofAgriculture/NationalInstituteofFood
versal methyl donor. The presence of METH implies that Por- andAgricultureHatchProject1004051(toA.S.K.andY.C.);theBiotechnol-
phyra takes up B from its abundant bacterial epiphytes (95), ogyandBiologicalSciencesResearchCouncil(BBSRCBB/1013164/1)ofthe
12
and we found that Porphyra, unlike other sequenced red algae, United Kingdom and European Union FP7 Marie Curie ITN Photo.Comm
317184(toA.G.S.andK.E.H.);the Officeof Biological and Environmental
encodesbothCOBTandCOBS.Theseenzymesareinvolvedin
Research of the US DOE (C.E.B.-H.); the Connecticut Sea Grant College
remodeling cyanobacterial-produced pseudocobalamin (101) to Program (R/A-38) andthe NOAA NationalMarine Aquaculture Initiative
8of10 | www.pnas.org/cgi/doi/10.1073/pnas.1703088114 Brawleyetal.
S
U
L
P
S
A
N
(C.Y.); the NIH MCB 1244593 (to H.V.G.); NSF and NIH Grants NSF-MCB NE/L013223/1 (to C.M.M.G. and Y.B.); NOAA Contract NA14OAR4170072 P
1412738, NIH P20GM103418, and NIH P20GM103638 (to B.J.S.C.O.); NSF (to S.H.B.); and The Great Barrier Reef Foundation, Australian Research
Graduate Research Fellowship under Grant 1247393 (to B.N.S.); the UK Council (DP150101875) and a University of Queensland Early Career Re-
NaturalEnvironmentResearchCouncilIOFPump-priming+schemeGrant searcherGrant(toC.X.C.).
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