Concluding Embryogenesis After Diaspora: Seed Germination in Illicium Parviflorum

Synopsis Albuminous seeds, dispersed with a minimally developed embryo surrounded by nutrient storage tissue, are pervasive across extinct and extant early diverging angiosperm lineages. Typically, seed ontogenic studies have focused on the time between fertilization and seed release, but in albuminous seeds, embryogenesis is incomplete at the time of seed dispersal. Here, I studied the morphological and nutritional relationships between the embryo and the endosperm after seed dispersal in Illicium parviflorum (Austrobaileyales). Seeds of I. parviflorum germinate over a period of three months. Different stages during the germination process were anatomically evaluated using a combination of histochemistry and immunocytochemistry. At dispersal, the seeds of Illicium contain a tiny achlorophyllous embryo with minimal histological differentiation, surrounded by copious amounts of lipo-protein globules stored in the endosperm within cell walls rich in un-esterified pectins. Six weeks later, the embryo expanded and differentiated the vascular tissues before the emergence of the radicle through the seed coat, as the stored lipids and proteins coalesced within cells. Six weeks later, the cotyledons contained starch and complex lipids intracellularly, and accumulated low-esterified pectins in their cell walls. The proteolipid-rich albuminous seeds of Illicium exemplify how woody angiosperms of the Austrobaileyales, Amborellales, and many magnoliids release seeds with high-energy storage compounds that are reprocessed by embryos that complete development during germination. Seedlings of these lineages thrive in the understory of tropical environments, which match with the predicted habitats where angiosperms evolved.


Introduction
More than half of the roughly 450 angiosperm families described to date store se e d nut rients in a fu l ly dev eloped embry o, and hence are exalb umino us ( Martin 1946 ).Most of these families belong to the eudicots ( Fo rb is et al. 2002 ).Upo n rele ase, t hese embryos are t ypic a l ly mature to break the se e d co at an d establis h an au to no mous se e d ling ( Lin kies et a l.2010 ).How ev er, in around half of extant m on o cots sp e cies ( Sre enivasu lu and Wobus 2013 ), in most m agnoliid s ( Frii s et al. 2015a( Frii s et al. , b , 2019 ) ), an d in th e b asa l g rade of ang iosperms that in clude Amb orell a , Nymphaeales, an d Aus tro baileyales (ANA), extant an d extin ct taxa release se e ds with an immature minute embryo surrounded by substant ia l nut rient-s tora ge tis s ue, which may be of materna l orig in (i .e., p erisp erm) or a product of fert i lizat ion (i .e., endosper m); f urt her a nd significa n t developmen t o f the emb ryo wi l l occur after se e d dispersa l, a lthough the deg re e o f emb ryo differen tia tion varies across taxa ( Grushv itzky 1967 ;Nikol aeva et al. 1985 ; Bas kin an d Ba skin 1998 ; Friedm a n a n d Bach e lier 2013 ; Povilus et a l. 2015 ;Losada et a l. 2017 ).D ur ing t he e arly ontogenic steps of the embryo, the cotyledons are hetero tro phic, a nd uptak e n utrien ts from the surrounding nu tri tive tis s ue (re vie wed in Povilus and Gehring 2022 ).In a l l al bumin ous se e ds, fol lowing dispersa l, the cotyle dons wit hin t he se e d s act a s fo o d tra nsf er orga ns, a n d som e nev er tran si tio n to n utrien t s tora ge (e.g ., Ny mphaeales: F riedman 2008 ; F rie dman et a l.2012 ; Frie dma n a nd Bach e lier 2013 ; Povilus et al. 2015Povilus et al. , 2018 ; ;Lu and Mag nani 2018 ;Da lziel l et a l. 2019 ; som e m embers of th e A nnon aceae: de Vogel 1982 ).In most alb umino us se e ds wit h endosper m, t he embryo uptakes n utrien ts from the endosperm unt i l th ey em er g e as ph otosynth etic, such as in Amb orell a an d in th e few Aus tro baileyales s tudied to date ( Chien et al. 2011 ;Fourcade et al. 2015 ;Fogli ani et al. 2017 ;Losad a et al. 2017 ).As a resu lt, a l bumin ous se e ds with a massive endosperm often disp lay pro lon g e d tempora l frames betwe en se e d dispersal from the m atern al sporophyte and seedling establis hm en t ( F ogliani et a l.2017 ; Vi l legente et al. 2017 ), but the sequence of events that occur during t his per iod are under studied .The alb umino us se e ds of the ANA grade offer an excellent system to bett er under st and t he lar g el y unexp lore d sp at iotempora l p a tterns of n utrien t mobiliza tio n fro m the endosperm toward a maturing embryo after se e d release ( Losada et al. 2017 ).
Al bumin ous se e ds are li kely a plesio mo rphic co ndi tio n o f flow erin g plan ts ( F o rb is et al. 2002 ), and the nature o f emb ryo-n ouris hing reserves accum ula ted in al bumin ous se e ds can be divided into two basic t ypes w i th a p redo minance o f ei t her st arch o r a co mb inatio n o f li p ids and pr oteins.Rar e ly do th ese two types of reserves cooccur in the same s tora ge tis s ues and accum ula te eit her in t h e en dosper m or in t he p erisp erm ( Friedma n a n d Bach e lier 2013 ).Intrace llula rly, sta rc h st orage occur s mainl y within p la stid s, wher eas pr ocessing of oil droplets inv olv es a biochemica l p at hway t hat goes t hrough t he pla stid s, the endoplasmic ret icu lum, and eventua l ly accumu lates in th e cytoplasm, an d som e steps are s hared wi th p rotein s ( Ohlrogg e and B rowse 1995 ; Herma n a nd La rkins 1999 ).While ma ny a ngios perm s pecies with albuminous se e ds store starch in the endosperm or the p erisp erm at disp er sal , f or exa mple, se e ds of g rasses ( Zhao et al. 2018 ), wo o dy memb ers of the ANA grade lineag es of flow erin g plants (Amborel la les and Aust rob ai lyea les), have se e d s th at ar e r ele ased wit h an endosperm lo ade d wi th li po-p rotein b o dies ( Fig. 1 ).These c haract er s have been reported in the families Amborell aceae (Amborell ales, Floyd and Fr iedman 2001 ), Tr imeniaceae (Aus tro ba ileyales, Friedma n a n d Bach e lier 2013 ), or Schis andrace ae (Aus tro baileyales, Hayashi et al. 1963a , b ;K ap il a nd Jala n 1964 ;P rakash 1998 ;Flo yd andF rie dman 2000 , 2001 ).The on ly except ion is the highly apo mo rphic mo notyp ic Aus tro baileyaceae, which p rod uce m a ssive se e ds wit h a st archy endosper m, though co mb ined wi th li p ids and p r oteins ( Endr ess 1980 ;Solnts e va 1981 ;Yamada et al. 2003 ;Losada et al. 2017 ).
Th e ear lies t angios perms were likely wo o dy, and prop agate d their se e ds in da rk a nd humid environments, which co rrespo nd wi th trop ical-like hab i tats ( Feild and Arens 2007 ).Examples of these specialized hab i tats can be found in living members of t he bas al ang iosperm g rade , suc h as the shrubb y Ambore lla , native to the t ropica l is lan d of New Ca le do nia, o r th e lian oid Austrob ail ey a , spe cifica l ly g rowing in the Aust ra lian t ropica l forest.The order Aus tro b ai leya les is the sister lineage to a l l euang ios perms (Ma gn oliids, m on ocots, and eudicots), composed by three families of wo o dy plants.Wit hin t he family Schis andrace ae, t he genus Ill icu m , conta ining a round 40 species, is composed of s hru bs native to shady t ropica l a nd wa rm tem pera te enviro nments o f the Asia n a n d Am erican contin ents, an d th e timin g of div ersificatio n o f the genus I l l ici u m co incided wi t h t hat bet ween Ny mphaea les and C h lorant hales ( Mor r is et al. 2007 ).Predis persal s ta ges of se e d deve lopm ent wer e pr eviously r eported ( Floyd and Friedman 2001 ), but pos tdis pers al infor mation remains uns tudied in mos t m embers of th e Schisan draceae an d the Aus tro baileyales.To better un derstan d th e embryogen esis of al bumin ous se e ds of the ANA grade and their evol u tio n in an giosperm s as a whole, w e used the s pecies I l l ici u m parvifloru m t o evaluat e the c han g es of bot h t h e embryo an d th e en dosper m dur ing se e d germination.

Plant material and seed collection
Plants o f I. parvi floru m wer e gr ow n in pl astic pots in the g re en houses of the Arnold Arbo retum o f H arvard Univ ersity, usin g high po rosi t y grow ing medium PRO-MIX (PremierTe ch Hort icu lture and Ag ricu ltur e Gr oup, Rivièr e-du-L oup, QC, C anad a).Indiv idu al plants r eached r epr oductive maturity under conditions of 25 ± 2 • C and rela tive h umidi ty (RH) o f 75%, and due to their under st o ry life hab i t, high s un expos ure was avo ided wi th a dar k n et between th e g re en house roof an d th e plants (av erag e ph otosynth et ic act ive radiat ion-PAR 100 μmol m -2 s -1 ).
Ma ture plan ts flowered t w ice per yea r, a nd ha nd pollination s w ere per for med at t he ant hesis st ag e usin g flowers from five different plants.While the f r uit set was t ypic a l ly low (the av erag e se e d set per fol licle was two ou t o f six), fro m flow erin g to f r uit disp ersal ab out threefour months elapse d.Swol len f r uits were col le cte d p rio r to opening, an d th e se e ds from th e de hisced exp losi ve follicles f urt her col le cte d for germinat ion.Thre e se e ds p er p ot were sown in PR O-MIX g enera l soi l ( n = 200 se e ds), ke ep ing the so il mo isture a t field ca pacity.Samples of five se e ds were col le cte d at different temporal intervals from diaspora (3, 6, and 11 weeks), and up to 15 wee ks wh en th e first cotyledons em er g ed in an epig eal fashion ( Fig. 2 ).

Seed processing for histochemistry
Fol lowing se e d col le ct ion, t he exotest a o f I. parvi florum se e d s wa s removed w ith a sc alpe l, an d th en fixed in 4% acrolein (Polyscien ces, In c.) in a modified P IP ES Fig. 1 Phylogeny of the genera from the ANA grade of angiosperms with emphasis in the reserves contained within the storage tissues of albuminous seeds, either in the endosperm (light blue) or the perisperm (pink).Reserves accumulated within the storage tissues go from lipids and proteins (yellow) to starch and proteins (dark blue), and associated papers that reported their embryogeny, either before or after seed r elease.Phylogen y and data obtained from Stevens, 2001 onwards.
buffer adjust ed t o pH 6.8 (50mM P IP ES and 1mM MgSO 4 fro m BDH, Lo ndo n, UK; 5mM EGTA from Resea rch Orga nics, In c., Cleve lan d, O hio, USA).Th ey were th en de hydrated with a n etha nol series up to 100% ethanol, infilt rate d and embe dde d in gl yco l methacrylate resin (JB-4 Po l ysciences, War r in gton, Penn sylvania, USA), and se ct ione d in 4 μm-thick ribbons using glass knives mounted on a rotary microtome (Microm HM360 from Th erm o Fis h er Scient ific, Wa ltham, Mass achusetts, USA).Ser ia l se ct ion s w ere moun ted on to s lides, stain ed with a p erio dic acid-Schiff 's reaction (PAS) t o det ect inso lub le carbo hydra tes, and coun terst ained wit h 0.01% aqueous to luidine b lue ( Feder and O' Br ien 1968 ).Furt her more, se ct ion s w ere stained with 0.25% Na ph t ol B lue B lac k in 1% acetic acid (ABB) t o stain proteins ( Fis h er 1968 ).

Immunolocalization of low methyl esterified pectins
The g erminatin g se e ds o f I. parvi floru m wer e col le cte d at th e deve lopm ent al st ages descr i bed an d , aft er removing the exotesta, fixed in 4% pa ra f orm aldehyde ( Solís et al. 2008 )   was applied for 1 h, then was h ed in 1XPBS, and incub ate d again in the dark for 45 min with the 1/25 PBS dil u ted seco nda ry a ntib o dy, an ti-ra t con juga ted with Alexa 531 and 488 fluo r ochr omes r espe ct i vel y (Thermo Fis h er, Waltham, Massach usetts, USA).F ol lowing thre e was h es in PBS, the se ct ion s w ere count er st ained wit h calco fluo r whi t e t o expose cel l wa l ls ( Hugh es an d McCu l ly 1975 ), and fina l ly mounte d in ProLong Gold An tifade reagen t (Th erm o Fis h er) p rio r to microscop ic observation.

Digital imaging
Images of fresh se e ds were taken using a Discovery A xioVi sion di sse ct ing microscop e (C arl Zeiss, Ob erko c hen, Germany).Phot ographs of microt ome se ct ion s w ere taken usin g a co mb inatio n o f b right field a nd D I C, wi t h t he Zei ss A xio Im ager Z2 microscope e quippe d with Zeiss High Resolution AxioCam digital ca meras (Ca rl Zeiss, Oberk ochen, Germa ny).Alexa 488 wa s vi sua lize d with a Zeiss LSM700 Confocal Microsco pe, equip ped with an AxioCam 512 camera, ill uminated wi th a fluo rescen ce Lum en cor SOLA-365-S E light, Chro ma fil ter set 49002, exci tatio n BP 470/40, beams plitter T 495, emis sion BP 525/50.Figures invo l ving whole se e ds in a l l deve lopm ent al st ages are t he result of photo mer g es of mu lt ip le indi v idu al images treated wit h t he Adobe Cre ative Suite 5 (Ado be Sys tems, San Jose, California, USA).

Postdispersal seed onto g eny of I. parviflorum
External morphology of I. parviflorum seeds during germination S ix weeks aft er se e d sow ing , the micropyl ar endosperm, pressed by the enlar g em ent of th e embryo, pus h ed an d broke the hilum in I. p arviflorum ( Fig. 2 A).Radicle emer g ence took place 3 weeks later (9 weeks after disper sal , Fig . 2 B), w i thou t any do rmancy r equir ements, b ut kep t the two co tyle dons g rowing into two white flat lobes at the expense of th e en dosper m inside t he se e d.As the hyp o cot yl elongated ( Fig . 2 C), the cot yledons con tin ued their elo ngatio n inside the se e d co a t un t i l just before they emer g ed from the soi l, fu l ly g re ene d, in an ep igeal fashio n around fifte en we eks fol lowing dispersa l ( Fig. 2 D).
Internal seed morpho-anatomy of I. parviflorum during germination At the se e d dispersa l stage, a h eart s hap ed embryo o ccu p ie d a minima l p art of the se e d, displaye d a protoderm, but no signs of protovascular tis s ue, and the majo ri ty o f the se e d was occu p ied by the endosperm, devo id o f star ch r eserv es ( Fig. 3 A), but stained inten sely fo r p roteins and li p ids ( Fig. 3 B).Six weeks later, the embryo exp ande d inside the se e d, pressing bot h t he micropyla r a nd cha laza l endosper m are a ( Fig. 3 C), where t he st a ining f o r p roteins was mo r e intense ar oun d th e embryo ( Fig. 3 D), while the vascu lar bund les different iate d.Elev en w eeks aft er disper sal , the root exited the se e d, th e embryo e longated th e hyp o co tyl and co tyledo ns occu p ied a lar g er area of th e en dosperm ( Fig. 3 E), which con tin ued to stain intensely for proteins toward the se e d periphery ( Fig. 3

F).
Polysaccharides in the embryonic tissues D et ai le d eva luat ions of the endosperm and embryo tissues revea le d tha t, a t disper sal , th e en dosperm ce lls con tained n umero us cyto plasmic glob ular b o dies and con serv ed their nuclei ( Fig. 4 A).In contrast, the area  aroun d th e embryo was devo id o f cell co n ten ts, but ma inta in ed th e integrity of th e ce l l wa l ls ( Fig. 4 B).Six weeks later, the endosperm cell con ten ts coalesced, pus hing th e nuc lei t oward t he edges of t h e ce lls, an d th e intercel lu lar sp aces en lar g ed, where in so lub le po l ysaccharides accum ula ted ( Fig. 4 C).The embryo surrounding area displayed a high concen tra tio n o f sugars, wi th co mp resse d cel l wa l l s, a s coty ledons acc um ula t ed starc h ( Fig .4 D).At l a ter germina tion s ta ges, th e en dosperm cell con ten ts disin tegra ted, their n uclei were no longer det ectable , an d th e ce l l wa l ls thinne d dow n ( Fig .4 E).Co nco mi t ant ly, t he cotyledons displayed differen tia tion of two external dermal layers, and the internal one accum ula ted tannins ( Fig. 4 F).Lipids and proteins in the embryonic tissues Th e absen ce o f p rotein st ain in t h e ce l l wa l ls cont raste d wit h intense st aining of t he globular con ten ts of the endosperm cells ( Fig. 5 A).Protein s w er e not pr esent in the embry o surroundin g area ( Fig. 5 B).Six w eeks after sowin g, protein s from the endosperm co a lesce d onto lar g er vesic les, and nuc lei were st i l l v isible ( Fig .5 C).The expandin g embry o contacte d dire ct ly t he co a lesce d proteins ( Fig. 5 D).At later s ta g es, the protein s broke down into sma l ler frag men ts tha t s tained acros s cel l wa l ls ( Fig. 5 E).The differen tia ted cotyledons s h owed m ost protein st aining in t h eir ce ll nuc lei , m ore s light ly in t he cytoplasm, an d compoun ds t hat re acted faint ly fo r p roteins accum ula ted under the epidermis ( Fig. 5 F).
The vesicles of t he endosper m cells at seed dispers al f urt her re acted for lipids, except in th e ce l l wa l ls ( Fig. 6 A).Sma l ler li p idic v esicles w er e pr esent in the embry o surroundin g area ( Fig. 6 B). S imilar t o prot eins, the li p ids o f th e en dosperm co a lesce d p rio r to se e d ling emer g ence ( Fig. 6 C), and the subdermal layer of the coty ledons acc um ula ted s ubs t ances t hat f urt her re acted fo r li p ids ( Fig. 6 D).
Pectin epitopes in the cell walls of embryonic tissues Low methyl esterified pectins pervaded the cell walls of th e mature en dosperm in I. p arviflorum at th e se e d dispers al st age ( Fig. 7 A).In contrast, the immature emb ryo nic cel l wa l ls co ntain li ttle o f these pe ct ins ( Fig. 7 B).During the init ia l s ta ges of embryo elongation, pe ct ins of the endosperm cel l wa l ls hyperaccumu late d in the apoplastic spaces ( Fig. 7 C), in cluding th e collapse d cel ls around the embry o surroundin g a rea, a nd were co nco mi t ant ly det ect ed in the epidermal tis s ues of the embryo ( Fig. 7 D).At later s ta ges of se e d germinat ion, pe ct ins vanis h ed from th e interce l lu lar sp aces of th e en dosperm ( Fig. 7 E), but the neo fo rme d wa l ls of the embryo accum ula ted p ectins, esp ecially in the protovascul ature ( Fig .7 F).

Discussion
The co mplete emb ry og enesis of alb umino us se e ds of t he bas al angiosper m grade genera such as I l l ici u m contribut es t o a bett er re const ruct ion of the developmenta l p athways of embryonic tis s ues in the earliest angiosper m line ages.

Delayed embryo maturation in albuminous seeds of the ANA grade
Embry og enesis of I. p arviflorum is completed between three and four months after se e d dispersa l, when disconne cte d from th e m oth er plant (see also Thien et al. 1983 ;O lsen an d Ruter 2001 ).Th e temporal fram e that el apses bet we en se e d re lease an d test a bre ak t akes more t han 2 mont h s in w o o dy memb er s of the ANA grade .Th us, Ambor ella takes about 3 months for testa break ( Fourcade et al. 2015 ), and within Aus tro baileyales, this temporal frame varies from 2 months of studied species o f Sc hisandra ( Chien et al . 2011 ;Hayashi 1963a , b ), t o the unusua l l y pro lon g ed 12 months of Austrob ail ey a ( Losada et al. 2017 ).In cont rast, the se e ds of the aquatic h erbs Nymphae la les typica l l y disp lay p erio ds of germinatio n o f days in absence o f p hysio log ica l dormancy

Completion of embryo g enesis during germination of albuminous seeds
Dur ing t he init ia l s ta ges o f I. parvi florum se e d germinatio n, p rio r to radicle emer g ence, the sequence of events in the endosperm invo l ves co a lescence of int racel lu la r proteins a nd lipids, a n d th eir re lease to th e apoplas tic s pace around the elon gatin g embry o.The heter otr ophic c haract er o f the cotyledo ns i s vi sua l ly displayed by their achloro phyllo u s n ature toget her wit h th e absen ce of a defin e d cut icle on their surface.Mobi lizat ion of n utrien ts stored in the endosperm is conco mi tant wi th the b iosynthesis o f starch in the cotyledons an d th e g radua l different iat io n o f tis s ues invo l ved in trans port, s uch a s the va scu lar bund les.This s ta ge is co mmo n to a l l a lb umino us se e ds, but whi le some taxa hav e cotyledon s as haustorial-fo o dsto ring o rgans, such as a l l Nymphaea les ( Frie dman 2008 ; Frie dman et al. in cluding th e m on otypic Aus tro baileyaceae , whic h displaye d not on ly li p ids and p roteins in th e en dosperm, but also starch deposits ( Slonts e va 1981 ;Losada et al. 2017 ).In species with a starchy p erisp erm as the main s tora ge tis s ue, li p ids and protein s w ere dete cte d in their high ly re duce d endos perm, s uch as in Nu p ha r ( Flo yd a nd Friedma n 2001 ).P re vious e va luat ion of lipids in the se e ds of a ran g e of an gios perm s pecies (n ot in cluding t he ANA grade), reve aled a tigh t associa tion between higher se e d oi l con ten t and b oth wo o din ess an d adaptation to dar kn ess ( Levin 1974 ;Sanyal and Decoq 2016 ).The genus ll ici u m and other members of the Aus tro baile yales are compos ed of species t hat t hr ive in t h e un dersto ry, ad justin g w ell to this co rrelatio n.In addi tio n, the ea rliest a n giosperm s w ere likely w o o d y and evo l ved in stable env ironments w ith low light and high humidity ( Fink elstein a nd Grubb, 2002 ;Feild and Arens 2007 ), point ing to a l bumin ous-oi ly se e ds as co mmo n in these scenarios ( Floyd and Friedman 2000 ).
The question remains on wh at i s the benefit, if any, o f sto ring li p ids and p roteins.W hile p r oteins ar e universa l ly store d within se e ds, an d provide th e materials for the enzymatic machinery tha t ca talyzes biosynthet ic p a thways ( Altsh u l et a l . 1966 ;Purkrt ova et al . 2008 ;Yan et al., 2014 ), li p ids a nd ca rbohydrates provide the energy, t ypic a l ly in t he for m of ATP ( Q uett ier an d Eastm on d 2009 ; Bat es et al .2013 ; O hlrogge an d Bro wse 1995 ).Ho wever, li p id s appear a s more efficient p acking compounds, g iven that oi ls provide a higher energy per unit m a ss comp are d w ith c arb ohydrates ( L evin 1974 ).Addi tio nally, li p ids would prevent the dehydratio n o f seeds mo re efficient ly t han st arc h ( Westgat e 1994 ).St arch, on t he ot her hand, appe ars to be more readi ly avai lable, and this cou ld be relate d with higher germina tion ra t es, suc h as thos e obs erved in s ome herb ace ous plants ( Xu et al. 2014 ;Zhao et al., 2018 ).Th e hydroph ob ici ty o f the li p ids co ntained wi t hin t he endosperm of alb umino us se e ds might increase se e d lon g ev it y under scenarios of high humidity ( Ellis et al. 1989 )

Conclusion
Se e d dispersa l a ffe cts spe cies dist ribut ion and community assemb l y, but does not mean the beginning of au-to no mous life in a l l ang iosperms ( Ing ram 2020 ).Whi le the essent ia l tool ki t fo r an in depen dent life is built in the emb ryos o f exal bumin ous plan ts a t th e tim e of se e d release, in al bumin ous plants from t he bas a l g rade of ang iosperms (and li ke ly oth er taxa with un derdeve loped embryos at se e d maturi ty), co mplete tis s ue differentiat ion is fu l ly depen dent on en dosp erm (Amb orel la les and Aus tro baileyales) or peris per m (Nymphae ales) p rovisio nin g.Surmountin g th e boun daries of a se e d is a major step in the life cycle of a new sporophyt e , bu t d ur ing t he init ia l steps of an giosperm ev ol u tio n, emb ryo au to no my was achie ved s ep arate d from the m oth er plant aided by a lon g-liv ed en dosperm.Th e se e ds of I. p arviflorum i l lust rate how living members of ea rly bra nches of flow erin g plan ts con tribut e t o a better un derstan ding th e trem en dous diversificatio n o f seed m orph o-physiolog ica l t ra its across a n giosperm s from ancest ra l immature embryos.

Funding
Thi s work h a s be en p art ia l ly funde d by a Put nam Scholarshi p fro m t he Ar nold Arbo retum o f Ha rva rd University, an d th e Spanis h Ministry of Science and I nno vation, t hrough t h e Agen cia Est at a l de Invest igación, wi th a r efer ence PID2021-129074OB-I00.
in 0.1M P hos p hate B uffer Saline (PBS).All fixe d spe cimens were rinse d in PBS, an d de hydrated through a g rade d series of acetone up to 100%.They were then embe dde d in Te chnovit 8100 (Ele ct ron Microscopy Scien ces, USA), an d se ct ione d at 4 μm-thick ribbons as described a bov e. Se ct ion s w ere placed on Superfrost s lides, was h ed wi th PBS fo r 5 min, and p re in cu b ate d with 5% bovine serum albumin (BSA) for five m ore min.Th en, we used th e m on oclona l ant ib o dy JIM5 ( Knox 1997 ), which detects low methyl esterifie d pe ct in ep i topes.Th e un dil u ted p rima ry a ntib o dy

Fig. 2
Fig. 2 External morphology of I. parviflorum seeds during germination.(A) Germinated seed six weeks after dispersal/sowing (6w), showing breakage of the testa at the edge of the hilum, and protrusion of the micropylar endosperm.(B) Eleven weeks after dispersal (11w), the radicle breaks the endosperm cap.(C) The hypocotyl continues to elongate 13 weeks after dispersal (13w) but kept an achlorotic nature.(D) Fifteen weeks after dispersal (15w), the greening of the embryo was concomitant with the proliferation of cotyledons in an epigeal fashion (composite image).Cotyl, cotyledons; End, endosperm; Exo, exotesta; Hil, hilum; Hyp, hypocotyl.Scale bars: 2mm.

Fig. 3
Fig. 3 Internal morphology of I. parviflorum seeds during germination.(A) Fully expanded endosperm at the time of seed release (0w), combined with a tiny heart shaped embryo.B. Intense protein staining in both the endosperm and the embryo, except in the embryo surrounding area.(C) Six weeks later (6w), the embryo elongated within the seed, developed vascular bundles, and broke the exotesta, yet kept a layer of endosperm around the root tip.(D) At this stage, the endosperm displayed a more intense color in the areas around the expansion of the embryo.(E) Eleven weeks after seed release (11w), the cotyledons of the embryo occupied part of the endosperm area, and concomitantly, a big portion of the endosperm was depleted.F. Protein staining expanded to peripheral areas of the seed.Compositions of multiple images w er e obtained from 4 μm resin sections, stained with PAS for soluble carbohydrates (pink color), and counterstained with toluidine blue for general structure (blue color).Emb, embryo; end, endosperm.Scale bars: 2mm.

Fig. 4
Fig. 4 Polysaccharides in the endosperm and the embryo during seed germination of I. parviflorum .(A) At the time of seed dispersal (0w), the endosperm is composed of nucleated storage cells (red ar ro wheads).(B) In contrast, the area of the endosperm surrounding the embryo was devoid of cytoplasmic material, yet keeping small nuclei (red arrowhead).(C) Six weeks after dispersal (6w), the cellular contents of the endosperm coalesced, and pushed the nuclei against cell walls (red arrowheads).(D) The embryo surrounding area was a tightly packed mass of cell walls (white ar ro ws), as starch accum ulated in the developing cotyledons (black ar ro ws).(E) Elev en w eeks after seed r elease, the contents of the endosperm cells disintegrated and cell walls thinned down.F. The embryo differentiated the epidermal and subepidermal tissues.4 μm resin sections stained with PAS for insoluble polysaccharides (pink), counterstained with toluidine blue for general structure (blue color).Cotyl, cotyledon; emb, embryo; end, endosperm; esa, embry o-surrounding ar ea.Scale bars: 20 μm.

Fig. 5
Fig. 5 Proteins in the endosperm and the embryo during seed germination of I. parviflorum .(A) At the time of seed dispersal (0w), the endosperm is composed of condensed cells with protein bodies (ar ro ws).(B) While the embryo sur rounding area of the endosperm is devoid of proteins, the cotyledons contained numerous small protein bodies.(C) Six weeks after dispersal (6w), larger protein bodies within endosperm cells suggested coalescence, contained in nucleated cells (red arrowhead).(D) The embryo surrounding area showed contact of endosperm proteins with the expanding embryo (the dotted line depicts the boundary between the endosperm and the embryo).(E) Eleven weeks after seed release, proteins broke down into small pieces that traversed cell walls.(F) In the differentiated embryo, proteins concentrated in the nuclei, and the sub-epidermal tissues displayed cell compounds with birefringence (ar ro ws).A total of 4 μm resin sections stained with ABB for general proteins (dark blue) imaged with differential interface contrast (DIC).Cotyl, cotyledon; emb, embryo; end, endosperm; esa, embry o-surrounding ar ea.Scale bars: 20 μm.

Fig. 6
Fig. 6 Lipids in the endosperm and the embryo during seed germination of I. parviflorum .(A) The endosperm was filled with lipid globules at the time of seed release.(B) The embryo surrounding area contained some remnants of lipids compared with other areas of the endosperm.(C) Lipids within endosperm cells coalesced and filled most of the areas in the endosperm cells.(D) The cotyledons differentiated a subdermal layer that reacted for lipids at germination.A total of 4 μm resin sections stained with auramine O for lipids (green).Cotyl, cotyledon; end, endosperm; esa, embryo-surrounding area.Scale bars: A, C, 20 μm; B, D, 100 μm.

Fig. 7
Fig. 7 Immunolocalization of low methyl-esterfied pectins recognized by the JIM5 mAb in the endosperm and the embryo of I. parviflorum during seed germination.(A) At the time of dispersal (0w), the endosperm cell walls contained unesterified pectin epitopes (white ar ro ws).(B) In contrast, the developing embryo did not accumulate pectins that conjugate with JIM5 in the cell walls.(C) Six weeks after dispersal (6w), the pectin epitopes hyper accumulated in the apoplastic spaces of the endosperm (white ar ro ws).(D) In the embryo surrounding area, as endosperm cells collapsed, pectin epitopes w er e r eleased to the apoplastic space, and localized in the epidermal layers of the developing embryo (white ar ro ws).(E) Tw elv e w eeks after diaspora (12w), pectin epitopes vanished from the endosperm cell walls (white ar ro w).(F) In the embry o, how ev er, the cell walls of the cotyledons accumulated high amounts of pectins (white arrows), most remarkably in the proto-vasculature.Illicium parviflorum seeds immunolocalized with JIM5 monoclonal antibody linked to a FITC-labelled secondary antibody (green), counterstained with calcofluor white for cellulose (blue).Cotyl, cotyledon; end, endosperm; emb, embryo; esa, embry o-surrounding ar ea.Scale bars: 20 μm.
2012 ; Friedma n a n d Bach e lier 2013 ; Povi lus et a l.2015 , 2018 ; Lu and Magnani 2018 ; Da lziel l et al. 2019 ), and som e m embers of th e Ann onaceae fami ly (Mag nolia les, de Voge l 1982 ), term ed as cryp toco t yl ar se e d germinat ion, most a lb umino us se e ds t ransit ion to g re ening and th eir cotyledons em erge, nam ed phan erocot yl ar se e d ger mination ( Finneset h et al. 1998 ).In g erminatin g I l lici u m se e ds, radicle emer g ence o ccurs b efo re the b iosynthesis o f p rote ct ive materia ls such as suberin-like compoun ds in th e su b-epiderma l t is s ues, an d th e accumulatio n o f pe ct ins in th e ce l l wa l ls of the cotyle dons whi le g re ening occurs.The putative uptake of pe ct ins from th e en dosperm ce l l wa l ls by th e deve lo ping co tyledons h a s b een rep orted in m ode l species with al bumin ous se e ds, but so far, ther e ar e no repo rts o f their localization across members of the ANA grade.Most works on the pe ct ins from the endosperm come fro m p redispers al st ages of al bumin ous se e ds of herb ace ous plants such a s gra sses [ Oryza ( Pa lmer et a l.2015 ), Hord eum ( Se lvig et al. 1986 ), Triticum ( Chateigner-Boutin et al. 2014 ), or Brachyp o d i u m ( Gui l lon et a l.2011 )], or exa l bumin ous se e ds such as Arabi d o psis ( Cruz-Valderra ma et al. 2018 ) an d Ni coti ana ( Lee et al. 2013 ).These works pointed to a nu tri tio nal role of the pectins from the endos perm, s uppose d ly r epr ocess ed into ne w emb ryo nic cel l wa l ls ( Lu a nd Magna ni 2018 ; St einbrec h er an d Leu bn er-Mezer 2018 ).Yet, a handful of works have explored the presence of pe ct ins in the embry og enesis of w o o dy organisms with al bumin ous se e ds, such as Q uercu s ( L op es et a l.2016 ; Costa et a l.2019 ).O ur work s ugges ts a tropic role of pe ct in s durin g the tran si tio n fro m heter otr ophy t o aut o tro phy (i .e., g re ening an d em er g ence of cotyledons), w hich occ urs w hen se e ds are sep arate d from the m oth er plant.Oily endosperms in the albuminous seeds of Austrobaile y ales I n I. pa rvi florum , the co mpounds sto red wi thin cells of the endosperm are li p ids and p rotein s, and w e not ice d that this association is true for most wo o dy members of Aus tro baile yales ( Humphre y 1896 ; Floyd and F riedman 2000 , 2001 ; F riedma n a n d Bach e lier 2013 ), , w hereas pec tins from the endosperm cell walls might play the role on se e d hume ctat io n d uring embryo act ivat ion.Whi le nut rient mobi lizat ion toward a growin g embry o h a s be en thorough ly documente d in herb ace ous lineages of the ANA grade with starchy alb umino us se e ds (Nymphaea les: Frie dman et a l.2012 ; Povi lus et a l.2015 ), m ore wor ks are ne e de d for a thorough re const ruct io n o f the se e d germinat ion t raj e ctories in al bumin ous se e ds of wo o dy lineages ( L osada et al. 2017 ; Fig. 1 ).