Accumulation of Seed Storage Proteins and the Taxonomy of Poaceae

8/3/2019 Accumulation of Seed Storage Proteins and the Taxonomy of Poaceae

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P1. Syst. Evol. 206:243-257 (1997) PlantSystematicsandEvolution Springer-Verlag 1997Printed in Austria

Accumulation of seed storage proteins and thetaxonomy of Poaceae*LIXUE CHEN, HILDE FISCHER, and UWE JENSENReceived September 19, 1996; in revised version December 17, 1996

Key words: Poaceae. - Seed storage proteins.Abstract: The accumulation of specific seed proteins is a taxonomically valuable featureand can be u sed to add itionally characterize plant taxa. To date, m ainly crop proteins havebeen analysed in the Poaceae. In this investigation seed proteins from 147 species werescreened with emp hasis on legumin-like proteins and prolamins. The groups resulting fromevaluation of the protein profiles correspond with well-known subfamilies and tribes.Panicoideae are clearly separated from Pooideae. Within Pooideae, th e Bromeae plusTriticeae tribes revealed obvious similarities. Lolium, Festuca and Vulpia, generallyincluded in the tribe Festuceae, revealed a protein profile similar to the profile of theBromeae/Trit iceae. Legum in-like proteins are a ccumulated abundantly in Bambusoideaeand Pooideae except Bromeae/Trit iceae, however, only the species included in theAveninae subtribe produce "soluble"(globulin-type) legumins as already known fromAvena sativa.

T h e P oaceae are one of the l a rges t angiosperm fami l i es wi th ca . 10000 spec ies .T hey a re p robab l y a m o nophy l e t i c g roup (DOYLE& al . 1992) and clearly separatedfrom re l a t ed m ono coty ledo n fami l i es by the i r redu ced f lowers a r ranged in sp ike le t sas wel l as by the s t a rchy endosperm and an embryo wi th a scu te l lum-l ikeco t y l edon . T oge t he r w i t h t he C y p e r a c e a e and re l a t ed monocot fami l i es they arecharac ter i zed by the "win d-po l l ina t ion syn dro me " (DAHLGREN 1983) . S evera lspec ies a re eco logica l ly and economica l ly impor t an t . Grass l ands occupy a th i rd ofthe lan d's surfac e (SCHANTZ 1954), an d grasse s are th e m ain f eed ing reso urces forsavannah an imal s and l ives tock . Cerea l c rops (wheat , rye , r i ce , maize , mi l l e t ) a ree s sent ia l food co m pon en t s fo r hum ans .

Al tho ugh no genera l ag reem ent has been achieved for the main c l ass i f ica t ion ofthe fam ily [wi th a rang e o f only tw o subfa mil ies (TZVELEV 1989) up to 13subfami l i es (CARO 1982)] , usua l ly the subfami l i es B ambuso ideae , P oo ideae ,Cen to theco ideae , A rund ino ideae , Ch lor ido ideae , and P anico ideae a re r ecogn i zed

* De dic ate d to emer. Univ.-Prof. Dr FRmORICHEHRENDORWRon the occasion of his 70thbirthday


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244 L. CHEN & al.:(STEBBINS CRAMPTON 1961, CLAYTON& RENVOIZE 1986) . W ith in sub fam il ies , th es e pa r a t i on o f s om e t r i be s a nd s ub t r i be s i s s t i l l que s t i ona b l e . W i t h i n s ub f a m .Pooideae t h e t a x o n o m i c p o s i t i o n o f t h e Mel iceae a n d Stipeae i s unce r ta in(DAHLGREN & al. 19 85 , SOREN6 & al. 19 90 ). T h e Bromeae, in te rpre ted a s a sepa ra tetribe, e.g., by CLAYTON& RENVOIZE 1986) , a re in c lu de d in the "Triticum g r o u p " b yDAHLGREN & al . (19 85) , or b e lo ng to the t r ibe Festuceae (PRAT 1960) . Th ere la t ionship of seve ra l o the r t axa i s unc lea r , e .g . , o f Brachypodium, Catabrosa,Ehrharta, Koeleria, Phippsia, and Uniola (PRAT196 0, SORZN6 & al. 199 0).CLAYTON & RENVOIZE (198 6) dis cu sse d severa l im po rtan t c harac ters and us edt he m f o r ou t l i n i ng t he g ra s s phy l oge ny . T he t r op i c a l Bambusoideae are p e r c e i v e da s t he m o s t p r i m i t i ve e x t a n t g r as s e s a nd de s c e nda n t s o f t he e a r l y f o r e s t s a va nna hg r a s se s i n a s e m i - ope n e c o t one , w h i c h i s c o r r obo r a t e d by t h e r e s u lt s f r om ndhFs e que n c e a na l y s is (CLARK& am. 1995) . The oryzoid Bambusoideae a r e pe r c e i ve d a sl i nks t o t he t e m pe r a t e Pooideae. T he o t he r s ub f a m i l i e s , c ha r a c t e r i z e d by am e s o c o t y l , m i g h t h a v e a c o m m o n o r i g i n w i t h t h e Centothecoideae, i n c l u d i n gArundinoideae , Chlor idoideae , a n d Panicoideae a s i n d e p e n d e n t l y e v o l v e dde s c e nda n t s . W i t h i n t he s e t h r e e s ub f a m i l i e s t he C 4 pho t os yn t he s i s pa t hw a ye vo l v e d i n pa r a l le l e vo l u t i on .R e c e n t l y D N A d a t a h a v e b e e n s u c c e s s f u l l y u s e d i n g r a s s t a x o n o m y a n dp h y l o g e n y . A c h l o r o p la s t D N A i n v e r si o n h as b e e n d e t e c t ed i n d i c a ti n g t h em o n o p h y l y o f Poaceae (DOYLE & a l. 1992) . Th e subfam i ly s t ruc ture ou t l ined byCLAYTON & RENVOIZE( 1986) w a s l a r ge l y c on f i rm e d : I n t he ndhF s e que n c e a na l y s is(CLARK & a l. 1995) the neo t ropic a l he rbac eou s Bambusoideae t r ibes Anomo-chloeae a n d Streptochaeteae appea r a s s i s te r grou ps to the r e s t o f the f ami ly . A lso ,a s e pa r a t i on o f t he Poaceae i n to a " B O P " c l a d e (Bambusoideae p l us Oryzoids, andPooideae) a n d a " P A C C " c l a d e (Panicoideae, Arundinoideae, Chloridoideae,Centothecoideae) ha s be e n s uppo r t e d (DAVIS& SORENG 199 3: RF LP ; BARKER& al.1995: rbcL; CLARK & al. 199 5: ndhF). A n i n t e r e s t ing m o l e c u l a r a na ly s i s ba s e d onI T S s e que nc e s f r om nuc l e a r r D N A i nd i c a t e d a s i s t e r g r oup r e l a t i ons h i p be t w e e nPoeae/Aveneae a n d Bromeae/Tri t iceae (HslAO & a l. 1995) , M olecu la r da ta a l soi m p r ove d the kn ow l e dg e o f c e r t a in t r i bes ( e . g ., Oryzeae: DUVALL & al. 199 3;Poeae: SOR~NG & al . 1990 ) as we l l as infrag en er ic re la t io nsh ips (Bromus: PILLAY&HILu 1995; Festuca: DARBYSHIRE & WARWICK 19 92 ; Hordeum: BAUM & BAILEY1991; Poa: SORENG 1990 ; Brachypod ium: Sra & al. 199 3, CATAL~q & al. 199 5).A l s o s e e d p r o t e i n c ha r a c t e r s w e r e u s e d f o r va l ua b l e c on t r i bu t i ons t o t hes ys t e m a t i c s a nd phy l oge ny o f g r a s s e s . T he Poaceae s e e d p r o t e i n s , be i ng e s s e n t i a lnut r i en t s for humans , be long to a l l four pro te in groups c la s s i f i ed by OSBORNE(1924) , i . e . a lbumins (wa te r so luble ) , g lobul ins ( so luble in d i lu te sa l t so lu t ions ) ,p r o l a m i ns ( s o l ub l e i n a que ous e t ha no l ) , a nd g l u t e l i n s ( s o l ub l e i n d i l u t e a l ka l i o ra c id ) . T he y ha ve be e n s t ud i e d i n t e ns i ve l y e s pe c i a l l y i n c e r e a l s w he r e o f t e n p r o l a -m i n s a c c o u n t fo r > 5 0 % o f t h e t o ta l e n d o s p e r m p r o t e i n (SHOTWELL% LARKJNS 198 9) .

W h e a t (Triticum aestivum a nd c l o s e l y r e l a t e d c r op t a xa) , r ye (Secale cereale),a nd ba r l e y (Hordeum vulgare) be l ong t o t he t r i be Triticeae w i t h i n s ub f a m .Pooideae. T he i r s e e d p r o t e i n p r o f i le is dom i na t e d by p r o l a m i ns . T he y a r ec h a r a ct e ri z e d b y a n u n u s u a l a m i n o a c i d c o m p o s i t i o n w i th h i g h p r o l i n e a n d a m i d en i t r oge n , a nd l ow p r opo r t i ons o f ba s ic a n d a c i d i c a m i no a c i d s . T he r e a r e t h r e et ype s o f p r o l a m i ns , t he H M W , S - poo r , a nd S - r ic h p r o l a m i ns , w i t h a h i gh d i ve r s it y


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Seed storage proteins of Poaceae 245both on the gene an d protein level with in each group. H owever, also other proteinsare present in sm aller amoun ts, e .g. , a legum in-l ike protein cal led tr i t icin, detectedin Trit icum aest ivum up to 5% of the total se ed prote in am oun t (SINOH & al. 1991).Maize , sorghum and most of the o ther cu l t iva ted mi l le t spec ies be long tosubfam. Panicoideae. In seeds of these crop plants prolamins also dominate (cal ledzeins in Zea mays , and kafir ins in Sorghum). They are r ich in a lanine and leuc ine.In oats (Arena sativa: Pooideae, Aveneae) and r ice (Oryza sativa: Bambusoi-deae, Oryzeae), prolamins are a minor s torage compon ent account ing for less than15% of th e tota l se ed n itrog en (SHOTW~LL& al . 1988). In both species legum in-l ikestor age pro tein s (SHOTWELL & al. 1988, TAKAIWA & al. 1986) are the m ajo rcomponent . They are predominant ly g lobul ins in Arena , but glutel ins in Oryza.Despite their different solubil i ty they are homologous to the classical Fabaceaelegu m ins (ROBERT & al . 1985), a con dit ion w hich is wide spre ad am ongangiosperm s (ow n data, unpubl.) an d conifers (H ~E R & DANK 1996).Grass seed s torage pro te ins have a l ready been inves t iga ted sys temat ica l ly byseveral authors. The prolamin size diversi ty in Poaceae was s tudied by Hmv &ESEN (1988) using SD S-PAGE. Th ey found three ma jor sub fam ily groups, i .e . (1)Bambusoideae /Oryzo ideae , (2 ) Pooideae, and (3) Chlor ido ideae /Panico ideae /Arundinoideae , with the Pooideae prolam in pattern as the m ost variable in terms o fmolecular weight and subuni t he terogenei ty . From a sero logica l s tudy conductedby ESEN & HICU(1989), members of Pooideae and Panicoideae showed the greatestd iss imi lar i ty , wi th an in termedia te pos i t ion of Oryza (Oryzoideae), Den drocalam us(Bambusoideae) , and Eleus ine (Chlor ido ideae) . Fur the r immuno log ica l andelectrophoret ic s tudies concerned subfam. Arundinoideae (HILu & ESEN 1990),subfam. Chloridoideae (HILu & ESEN 1993), a nd the tribes Arist ideae (ESEN & HILU1991) and Triticeae (PELGER 1993). The antig enic relatio nsh ips betw een ba rley, ryeand wheat have been studied by FELSENSTErN& al. (1984), also confirming a lack ofc ros s - r eac t iv i t y be tween p ro l amins o f t he two sub fami l i e s Pooideae andPanicoideae. Antisera against crude protein extracts from the Z e a m a y s globul infractio n hav e be en u sed b y KHAVKIN & al. (1979). T he y disti ngu ishe d Coix andSorghum antigens from Zea, Euchlaena, and Tripsacum ant igens by sero logica ldifferences; globulins of the Andropogoneae clearly differed from their counter-parts in the Paniceae and were widely apart from globulins in other t r ibes ofsubfam. Pooideae. Re cen tly LUTI~ (1991) studied the proteins in several t r ibes ofthe Poaceae elec t rophore tica l ly and immu nologica l ly and found tha t Oryza sativa,Stipa viridula, Agrostis alba, Avena sativa, and Phalaris canariensis producelegumin-l ike proteins as the major storage protein fract ion.

In the present invest igat ion we report the screening results for major seedproteins from 147 Poaceae species and 103 genera: (1) The legum in-l ike proteinswi th a subuni t molecular weight (M.W.) of about 55kDa, charac ter ized bysuscept ib i l i ty to reduct ive c leavage which resul t s in the format ion of twopolypept ides of ca . 22 and 40kDa; (2) a pro lamin wi th a M.W. of 38kDa, and(3) the p ro lamin p rof ile cons ider ing especia l ly the h igh m olecular w eight pro te ins .This screening i s based on SDS-PAGE. Add i t ional ly ant isera agains t Avena sat ival egumin and the m ajor seed pro te in of Sorghum cernuum have been used for c ross-reac t iv i ty exper iments . Genera l ly only the accumula t ion of a pro te in has beennoticed; therefore the production of other proteins in unsignif icant amounts cannot


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2 4 6 L . C ~ N & a l. :t o t a ll y b e e x c l u d e d . T h e r e s u l t s e x t e n d o u r k n o w l e d g e o f t h e d i s tr i b u t i o n o f g r a s ss e e d p r o t e i n s b e y o n d t h e w e l l k n o w n c e r e a l p r o t e in s . C o r r e l a t i o n s w i t h d e f in e dt a x o n o m i c g r o u p s c o n t r i b u t e t o w a r d s a b e tt e r u n d e r s t a n d i n g o f t h e i n t r a f a m i l i a rp h y l o g e n e t i c r e l a t i o n s h i p s .Ma t er ia l a nd met ho dsPlant seeds . A b o u t 1 5 0 s p e c i e s o f 1 0 3 g e n e r a f r o m 1 9 t r i b e s h a v e b e e n s t u d i e d ( T a b l e 1 ) .T h e s e e d m a t e r i a l s w e r e o b t a i n e d f r o m t h e f o l l o w i n g B o t a n i c a l G a r d e n s , I n s t i t u t e s a n dC o m p a n i e s : B o t a n i c G a r d e n o f t h e U n i v e r s i t y o f C o p e n h a g e n , D e n m a r k ; R o y a l B o t a n i cG a r d e n s K e w , E n g l a n d ; J a r d i n B o t a n i q u e d e l ' U n i v e r s i t 6 d e L i r g e , B e l g i u m ; J a r d i nB o t a n i q u e d e B o r d e a u x , F r a n c e ; A n d r e K r o n e r , S w i t z e r l a n d ; I P K G a t e r s l e b e n , G e r m a n y ;B o t a n i s c h e r G a r t e n u n d Rhododendron-Park B r e m e n , G e r m a n y ; I n s t i t u t f t i r L a n d w i r t s c h a f t -l i c h e B o t a n i k , U n i v e r s i tf i t B o n n , G e r m a n y ; B o r nt r~ ig e r G m b H , O f f s t e i n , G e r m a n y ; F a .C o n r a d A p p e l , D a r m s t a d t , G e r m a n y ; R e s e a r c h I n s t i t u t e f o r F o r e s t r y , S i c h u a n , V R C h i n a .See ds o f s e vera l sp ec ies w ere co l l e c ted in na tu re b y D r PEDRO GERSTBER~ER,U n i v e r s i t y o fB a y r e u t h , G e r m a n y .Prote in extract ion and S D S - P A G E . S e e d s i n c l u d i n g th e s e e d c o a ts w e r e g r o u n d t op o w d e r , e x t r a c t e d in a b u f f e r (1 m l p e r 1 0 0 m g s e e d s) c o n t a i n i n g 1 0 r a M T r i s, 8 0 r a Mg l y c i n e , 1 M N a C 1 , p H 8 . 5 f o r 1 . 5 - 2 h a n d c e n t r i f u g a t e d a t 1 2 0 0 0 g f o r 1 5 r a i n . T h es u p e r n a t a n t ( a l b u m i n s a n d g l o b u li n s ) w a s r e m o v e d a n d s t o r e d a t - 2 0 C . T h e p e l l e t w a sr e e x t r a c te d t w i c e w i t h t h e s a m e b u f fe r , f o l lo w e d b y t h r e e e x t r a c ti o n s w i t h 7 0 % e t h a n o l t oo b t a i n t h e p r o l a r n i n f r a c t i o n ; f in a l l y a g l u t e l i n i c f r a c t i o n w a s g a i n e d b y o v e r n i g h t e x t r a c t i o nw i t h a b u f fe r c o n t a in i n g 6 5 m M T r i s / H C I , 1 0% ( v /v ) g l y c e ro l , 2 % ( w / v ) S D S , p H 6 . 8. A l lt h e s e f r a c ti o n s w e r e a n a l y s e d b y s o d i u m - d o d e c y l s u l f a t e p o l y a c r y l a m i d e g e l e l e c t ro p h o r e s i s( S D S - P A G E ) a s d e s c r i b e d i n JEN SEN & C ~ N ( 1 9 9 1) u s i n g n o n r e d u c i n g a n d r e d u c i n g ( 0 .2 %( w / v ) D T E ) c o n d i t io n s . S t a in i n g w a s p e r f o r m e d w i t h C o o m a s s i e B r i ll i an t B l u e a c c o r d i n gt o s t a n d a r d p r o c e d u r e s .

P repa ra t ion o f a nt ibo d ie s , immu no di f f us io n a nd i m m u n o b l o t t i n g . T h e l e g u m i n -l ik ep r o t e in , t h e m a j o r p r o t e i n o f o a t s e e d s w a s p u r i f i e d b y g e l f i lt r at io n c h r o m a t o g r a p h y ( A c AU l t r o g e l 3 4 , I B F ) u s i n g t h e g l o b u l i n e x t r a c t i o n b u f f e r . A n 3 8 k D a ~ - z e i n - l i k e g l u t e l i n ( s e eF i g . 1 ) f r o m Sorghum cernuum w a s s e p a r a t e d t h r o u g h S D S - P A G E , f o l l o w e d b y e l e c t r o -e l u t i o n ( B i o t r a p e q u i p m e n t , S c h l e i c h e r & S c h t i l l ) . T h e p r o c e d u r e s u s e d f o r a n t i b o d yp r o d u c t i o n h a v e b e e n d e s c r i b e d e l s e w h e r e (F IS CH E R & JE N SE N 1 9 9 2 ) . O u c h t e r l o n yi m u n o d i f f u s i o n t e s t s w e r e p e r f o r m e d e s s e n t i a l l y a c c o r d i n g t o F I SC H ER & JE NS EN ( 1 9 9 2 ) ,b u t u n d e r p a r t i a l l y d e n a t u r i n g c o n d i t i o n s w i t h 4 M u r e a i n t h e g e l s t o k e e p g l u t e l i n s i ns o l u ti o n . P r o te i n s w e r e t r a n s f e r r e d f r o m p o l y a c r y l a m i d e g e l s to n i t r o c e l lu l o s e m e m b r a n e sa c c o r d i n g t o K I aY sE -A N D ER SE N ( 1 9 8 4 ) ; b l o c k i n g a n d s u b s e q u e n t i n c u b a t i o n s w e r ep e r f o r m e d in " B l o t t o " ( 0 .0 2 M t ri s, 0 . 9 % ( w / v ) N a C I , 5 % ( w / v ) n o n - f a t d r y m i l k ) a t4 0 C . D e t e c t i o n o f c r o s s - re a c t i n g p r o t e i n s w a s a c h i e v e d b y t h e r e a c t i o n o f g o a t - a n t i- r a b b ith o r s e r a d i s h p e r o x i d a s e ( B i o R a d ) w i t h a s u b s tr a t e s o l u t i o n c o n t a i n i n g 0 .0 2 5 % ( w / v ) 3 , 3 -d i a m i n o b e n z i d i n e t e t r a h y d r o c h l o r i d e , 0 . 0 3 % ( w / v ) C O C 1 2, 0 . 0 3 % ( v / v ) H 2 0 2 .

N-terminal amino ac id sequencing . Echinochloa crus-galli p r o t e i n s ( g l u t e l i n f r a c t i o n )w e r e s e p a r a t e d b y S D S - P A G E u n d e r r e d u c i n g c o n d i t i o n s. T r a n s f e r o f p r o t e in s t o a P V D Fm e m b r a n e , s t a in i n g a n d a u t o m a t e d N - t e r m i n a l a m i n o a c id s e q u e n c i n g w a s d o n e a sdes cr ibe d in FISCHER & a l . (1995) .R e s u l t sT h e s p e c i es e x a m i n e d b e l o n g t o si x g e n e r a f r o m f o u r t r ib e s o f s u b f a m .B a m b u s o i d e a e , 6 5 g e n e r a f r o m e i g h t t ri b e s o f su b f a m . P o o i d e a e , t h r e e g e n e r a


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Seed s t o r ag e p ro t e i n s o f Poac e ae 247f r o m t w o t r ib e s o f s u b f a m . A r u n d i n o i d e a e , e i g h t g e n e r a f r o m f o u r t ri b e s o f s u b f a m .C h l o r i d o i d e a e a n d 1 6 g e n e r a o f t w o t r ib e s f r o m s u b f a m . P a n i c o i d e a e . T h e r e s u l t so f th e e l e c t r o p h o r e t i c a n a l y s e s a r e l is t e d in T a b l e 1 a n d s u m m a r i z e d i n T a b l e 2 .T h e y s h o w t h at t h e l e g u m i n s a r e a c c u m u l a t e d o n l y i n B a m b u s o i d e a e a n d P o o i d e a e( e x c e p t i n B r o m e a e / T r i t i c e a e a n d i n t h e L o l i u m g r o u p ) . C o r r e s p o n d i n g i m m u n o -Table 1 . The s eed p ro te in p rof i l e o f Poaceae . A l eg u mi n s " s o l u b l e" (o f t h e g l o b ul i n -t y p e ) ;B leg u mi n s " i n s o l u b l e" (o f t h e g l u te l i n- t y p e ): + + + h eav i l y accu mu l a t ed , + + accu mu -la ted , + p resen t , - no t v i sib le in PAG E of crude ex t rac t s , ? p resen ce doubt fu l ;C accumula t ion o f a 38 kDa M. W. p ro te in ; D pro lamins : S on ly low M. W. p ro te ins(up to 30 kDa) ; M add i t ional ly p ro te ins up to 45 kDa; L add i t ional ly h igh M. W. p ro te ins(M. W . ab o v e 4 5 k D a) p r e s en t an d o f t en accu mu l a t edT ax a A B C DS u b f a m . Bam buso i de aeB a m b u s e a e

Bashan i a f a rge s i i (CAMuS) KENt & YtFarges ia ni t ida (M~TF.) KENGPhy l l os tac hy s p ube sc e ns MAZEL ex LEgAtE

OryzeaeOryza sat iva L .

Ehrhar t e aeEhrhar ta calyc ina SM.E. longiflora SM.D i arrhe ne aeDiarrhena americana P. B.

S u b f a m . Pooi de aeNarde aeNardus s t r ic ta L .

St ipeaeAch nathe rum calamagros t i s P. B.M i l i um e f f u sum L .Oryzops is mi l iacea (L.) Ascii. & SCHWEtYF.St ipa e legant i ss ima LABmL.S. pennata agg.

Poe aeBr i za m e d i a L .Catabrosa aquatica (L.) P. B.Catapodium r igidum (L.) C. E. HuBB.Cynosurus cr i s ta tus L .Dac ty l i s g lomerata agg .Desmazer ia s icula (JACQ.)DUMORT.Echinar ia capi ta ta DESF.L am arc k i a aure a (L.) MOENCI~Poa chaix i i VmI..P. compressa L .P. nemoral i s agg .t3. palustris L.P. pratens is agg .

m

+ ++

m

m

m

m

?

++

+

+ + + - S+ + + - S+ + + - S

+ + + - S

+ + - S+ + + -

+ + + - S

+ + S+ + + - S+ + + - S+ + + - S+ + + - S+ + + - S

+ + ++ ++ ++ + ++ + ++ + ++ + ++ + ++ ++ ++ ++ ++ +

m

m

m

n

m

SSM

MSMMMMM


6/15

2 48T a b le 1 ( c o n t i n u e d )

L. CHEN a l . :

P u c c i n e l l i a d i s t a n s a g g .S e s I e r i a v a r i a a g g .C a s t e l l i a t u b e r u l a t a T I N .C u t a n d r a m a r i t i m a BENTH.F e s t u c a a r u n d i n a c e a SCHREB.E g i g a n t e a (L.) VILE.E o v i n a a g g .F . p r a t e n s i s HUBS.E r u b r a a g g .L o l i u m m u l t i f l o r u m L A M .L . p e r e n n e L .L . t e m u l e n t u m L .M i c r o p y r u m t e n e l l u m L .V u lp ia m e m b r a n a c e a (L . ) LK.V. myu ro s (L.) GMEL.

H a i n a r d e a eP a r a p h o l i s s t r i g o s a (DtrM.) C. E. HUBBARD

M e l i c e a eG l y c e r i a m a x i m a (HARTM.)HOLMS.M e l i c a c i l i a t a a g g .M . n u t a n s a g g .S c h i z a c h n e p u r p u r a s c e n s SWALE.

A v e n e a e - A v e n i n a eA i r a c a r y o p h y l l e a a g g .A r r h e n a t h e r u m e l a ti u s (L.) J. & C. PRESL.A v e n a b a r b a t a POTT ex LK.A . b r e v i s ROTHA . f a t u a L .A . s a t i v a L .A . s t r i g o s a SCHRES.A v e n e l l a f l e x u o s a (L.) PARE.A v e n o c h l o a a l b i n e r v i s (Bolss.) HOLUBA . p r a t e n s i s (L.) HOLUBC o r y n e p h o ru s c a n e s c e n s (L.) P. B.D e s c h a m p s i a c e s p i t o s a a g g .G a u d i n i a f r a g i l i s P . B .H e l i c t o t r i c h o n s e m p e r v i r e n s (VILE .) PrLG.H o l c u s l a n a t u s L .K o e l e r i a g l a u c a (Sc rmao.) DC.K . p y r a m i d a t a a g g .L o p h o c h l o a c r i s t a t a (L.) H,a~.R o s t r a r i a c r i s t a t a (L.) TSVELEVT r i s e t um f l a v e s c e n s a g g .

A v e n e a e - P h a l a r i d i n a eA n t h o x a n t h u m o d o r a t u m a g g .H i e r o c h l o e a u s t r a li s ( S c r i R ~ . ) R . & S c s .P h a l a r i s c a n a r i e n s i s L .

m

m

m

m

m

m

D

D

D

+ + + + + - S+ + + + - M+ + + ++ + + + - M+ + + ++ + + + - M+ + + + - M+ + + +++ + + + -+ + + + -+ + + + - S+ + + +++ + + + - M++ ++ -- + + + - S+ + + + - S+ + + + + -++ ++ -++ + + - S+ + + + - Sm

m

m

+ + ++ + + - S

- - - - L

- - - - L

- - L- - L- - - - L

- - - - L

- - L

- - L

- - L

- - S

- - L

- - L

+ + + - M

+ + ++ + + - S+ + + - S+ + + -

+ + + - M+ + - S+ + + -

T a x a A B C D


7/15

S e e d s t o r a ge p r o t e i ns o f Poaceae 24 9Ta b l e 1 ( c on t i nue d)Ta xa A B C DAveneae - Alopecurinae

Alopecurus bulbosus GOUANAmmophila arenaria a gg .Apera spica-venti (L. ) E B.Beckmannia eruciformis (L. ) HosT.B. syzigachne FERNACDCalamagrostis arundinacea (L.) ROTHC. epigeios (L.) ROTHChaetopogon fasciculatus (Ltc) HAYEKCinna arundinacea L.Gastridium australe E B.Lagurus ovatus L.Phleum phleoides (L.) KARSTENPolypogon monspeliensis (L.) DESEBromeaeBoissiera bromoides HOCHST. & S~U D.Bromus inermis LEYS.B. sterilis L.B. tectorum L.

TriticeaeAegilops geniculata ROTHA. tauschii C oss .A. ventricosa TAUSCHAgropyron caninum (L. ) E B.A. junceum a gg .Aneurolepidium sp .Asperella hystrix HUMB.Brachypodium sp .B. pinnatum a gg .B. sylvaticum (HuDs.) E B.Dasypyrum sp .Elymus arenarius L.E. dahuricus TURCZ.Eremopyrum sp .Hordelymus europaeus (L.) HAp,ZHordeum agriocrithon AABERGH. murinum a gg .H. vulgare L.Psathyrostachys juncea (FIscH.) NEvs~aSecale cereale L.Taeniatherum crinitum (ScHPaSB.)NEVS ~Triticum aestivum L.T. aethiopicum JAKUBZ.T. dicoccum SCHRAYK.T. durum DESET. monococcum L.T. spelta L.

+ ++ + - S- + + + - M+ ++ + - S+ ++ + - S+ ++ + - S- + + + - S- + + + - S- + + + - S- + + + - S- ++ - S+ ++ + - S- + + + - S

- + + + - S

??

LLLLLLLLLSSLLLLLLLLLLLLLLLLL


8/15

2 5 0T ab l e 1 ( co n t i n u ed )

L . C r ~ y & a l . :

T a x a A B C D

S u b f a m . ArundinoideaeArundineae

Molinia caerulea ag g .Notodanthonia buchananii ( H o o K . f. ) Z o zo v

MicraireaeAristida inaequiglumis DOMIN

S u b f a m . ChloridoideaePappophoreae

Enneapogon cenchroides (LIcnz .) HuB s .Eragrostideae- Uniolinae

Uniola latifolia Mlco_x.Eragrostideae - EleusinaeDactyloctenium aegypticum WINED.Eleusine coracana GAERTN.E. tristachya KtrNTH.Eragrostis pilosa (L.) P. B.E. tef (Zucc.) T. ROTTER

Eragrostideae - SporobolinaeSporobolus indicus R. BR.

Cynodonteae - ChloridinaeChloris distichophylla LAG.C. truncata R. BR.Cynodon dactylon (L.) PERS.

m

m

- S

- S

- S

+ Sm _ _ - - S

- - ? S- - - - - - S

? - - S

S u b f a m . PanicoideaePaniceae - SetariinaeBrachiaria vilIosa (LAM .) A. CAMUSEchinochIoa crus-gaUi (L . ) P . B .E. frumentacea L~.Panicum miliaceum L.Paspalum distichum L.Setaria glauca (L.) P. B.S. italica (L.) P. B.S. viridis (L.) P. B.

Paniceae - DigitariinaeDigitaria sanguinalis ScoP.

Paniceae - CenchrinaeCenchrus tribuloides L.Pennisetum americanum (L.) LEEKE

Andropogoneae - SorghinaeChrysopogon gryllus ( L .) T ~ .Sorghum bicolor (L.) MOENCHS. saccharatum (L.) MOENCH

Andropogoneae - AndropogoninaeAndropogon scoparius M l c n x .Botriochloa ischaemum (L.) KENG

m

m

_ q

m

D

- M

?- S- S

+ S+ S+ S+ S+ S+ S+ S+ S+ S+ S+ S+ S+ S+ S+ S? S


9/15

Seed s to rage pro te ins o f PoaceaeTable 1 (con t inued)

25 1

Taxa A B C DCymbopogon ambiguus A . CAMUS -- -- + S

Andr opogoneae- T r ips ac inaeTripsac um dactyloide s L. - - + SZea may s L. - - + S

Andropogoneae - CoicinaeCoix lacry ma-jo bi L. - - + S

Table 2 . Data f ro m the pro te in scree n ing of Table 1 , genera l ized for the subfam il ies o f thePoaceae. A Ac cum ulat ion of " so lub le" g lobul in ic (g lob) and "un so lub le" g lu te l in ic (g lu t)legumins. B Accum ulat ion of p ro lamins: S M. W. up to 30 kDa; M M . W. up to 45 kDa;L addi t ional ly p ro te ins wi th M. W . above 45 kDa. C Presenc e of the 38 kDa pro te inTax o n A B CBambusoideae glut S -Pooideae

Aveneae: mo st taxa g lu t S (M) -Aveneae: Aveninae glob S (M) -Poeae: Lolium group - L -Bromeae/Trit iceae - L -

Arundinoideae - S -Chloridoid eae - S -Panico ideae - S +

d i f f u s i o n e x p e r i m e n t s r e s u l t e d i n c r o s s - r e a c t i v i ty o f a n t i - A v e n a - l e g u m i n w i t h o t h e rP o o i d e a e l e g u m i n s ( n o m a t t e r w h e t h e r o f t h e g l o b u l i n o r g lu t e l in t y p e ) b u t n o t w i t hth e B a m b u s o i d e a e l e g u m i n s . T h e P o o i d e a e a r e c h a r a c t e r i z e d b y p r o la m i n s o f v e r yd i f f e r e n t M . W . , t h e p r o d u c t i o n o f h i g h M . W . p r o l a m i n s b e i n g r e s t r i c t e d t o t h eB r o m e a e / T r i t i c e a e i n c l u d i n g t h e L o l i u m g r o u p . T h e 3 8 k D a g l u t e li n is a c c u m u l a t e de x c l u s i v e l y i n P a n i c o i d e a e .

T h e r e s p e c t i v e p r o t e i n o f E c h i n o c h l o a c r u s - g a l l i ( 21 k D a i n it s r e d u c e d f o r m ,s e e a l s o F i g . 1 ) w a s a n a l y z e d a l s o b y N - t e r m i n a l a m i n o a c i d s e q u e n c i n g . T h i ss p e c i e s w a s c h o s e n f o r r e a s o n s o f a v a i l a b il i ty o f s u f f ic i e n t p l a n t m a t e r i a l . T h es e q u e n c e : V V I P Q C S I A A G A A T I Q Y i s s t r o n g l y s i m i l a r t o t h e p r e v i o u s l yr e p o r t e d ( O rT O BO N I & a l. 1 9 9 3 ) ~ - z e i n s f r o m m a i z e a n d t h e i r h o m o l o g u e s i nCoix a n d Sor ghum. T h e c ~ - z e i n s w h i c h u n t i l n o w c a n n o t b e r e l a t e d t o a n y o t h e rk n o w n s t o r a g e p r o t e i n (S HE WR Y 1 9 9 5 ) o b v i o u s l y a r e u n i q u e a n d d i a g n o s t i c t o t h et r i b e P a n i c o i d e a e . T h i s i s u n d e r l i n e d b y o u r i m m u n o b l o t t i n g e x p e r i m e n t s w i t ha n t i - S o r g h u m - z e i n , w h e r e c r o s s - r e a c t i o n s o c c u r o n l y w i t h p ro t e i n s o f p a n i c o i ds p e c i e s .


10/15

252 L. CHEN & al.:+ - + - + - +

940006700043000300002010014400

S I a I b ] c I d I sFig. 1. S DS-PAGE of Panicoideae seed glutelins using reducing (+) and non-reducing (- )conditions. The 38 kD a protein is ind icated by arrows. Observe also its 21 kDa subunitsund er reduc ed conditions. S Standard proteins (M.W . in Da), a Setaria glauca; b Panicummiliaceum; c Dig#a ria sanguinalis; d Echinoch loa frum enta cea

Discuss ionBam bus o i deae . The m ajor seed prote in i s an " inso luble" (g lu te l in- type) legumin,with i ts subunits revealing electrophoret ic propert ies s imilar to the legumin of thePooideae . However, missing cross-reactivi ty with the Avena l egumin indica tesconsidera ble differences in the mole cula r s tructure. Prolamins oc cur frequently, butthey are not a major component . The taxonomic pos i t ion of Ehrhar ta is dubious(e.g ., P ~ T 1960 ). CLAYTON RENVOIZE (198 6), CLAkI,: & al. (19 95 ) an d o the rsinclude this genus into the Bambusoideae , or it is believed to be a sister taxon o f theBam bus o i deae (I~LLOC~ & CAMPBELL 1986), or e xc lu de d fr om the Bam bus o i deaebranch at all by SODERSTROM& ELLIS (198 7) . DAI-ILGREN& al. (1985) link Ehrhar tawith the Arundinoideae , TZVZLEV (1989 ) wi th the Pooideae . Some anatomica lsimilari t ies also connect Ehrhar ta with the Aveninae genus Hel ic to t r i chon (HIIy1985). The two Ehrhar ta species tested by us differ from the other Bam bus o i deaespecies in producing globulin-type legumins in considerable amounts (Table 1) .This corresponds with subtr ibe Aveninae within the Pooideae , a l though miss ingserological cross-reactions with an antiserum against Avena l egumin do not favoura close relat ionship. Considering al l available protein data, the Ehrhar ta l eguminfeatures may be interpreted as an indicat ion of an early branching off of Ehrhar taf rom the bam busoid c lade , connected wi th a chang e f rom globul in-l ike to g lu te l in-l ike legumins; bu t further data are desirable to f inal ly jud ge the posi t ion of thisgenus.

Pooideae. The Pooideae , with the exclusion of the tr ibes Br om eae andTriticeae, genera l ly accumula te legumin- l ike pro te ins and produce par t icu lar lysmal l molecular weight pro lamins . The legumin- l ike pro te ins a re in most cases ofthe glutel in type. Only grasses w ithin the Aveneae-Aveninae produce g lobul in- type


11/15

Seed storage proteins of Poaceae 253l e g u m i n s i n l a r g e a m o u n t s . T h e p r e s e n c e o f g l o b u l i n -t y p e le g u m i n s h a s l o n g b e e nk n o w n f ro m Arena sativa ( BuR gEs s & a l. 1983 ), a nd i s no w d e m ons t r a t e d f o r m or eo r l e s s a l l m e m be r s o f s ub t r i be Aveninae. A n Arena sativa l e g u m i n a n t i s e r u mr e ve a l e d c r o s s r e a c t i v i t y w i t h t he l e gum i ns e x t r a c t e d f r om t he Aveninae spec ie s ,bu t a l s o w i t h t he g l u t e l i n - t ype l e gum i ns p r o t e in s f r om o t he r Pooideae. This i se x p l a i n a b l e b y s e q u e n c e h o m o l o g y o f b o t h t h e g l o b u l i n - t y p e a s w e l l a s t h eg l u t e li n - t y p e l e g u m i n s , w h i c h h a s b e e n d e m o n s t r a te d f o r Avena sativa ( g l obu l i n -t ype ) a nd Oryza sativa (glu te l in - typ e) (ROBERT & al . 1985) .T he p r o t e i n da t a f o r t he ge ne r a Festuca, Lolium, and Vulpia, g e n e r a l ly i n c l u d e di n t he Pooideae-Festuceae, do n o t f it t he c o m m on c ha r a c t e r is t ic s o f t he Poeae, ast h e y p r o d u c e p r o m i n e n t h i g h m o l e c u l a r w e i g h t p r o la m i n s , a n d d o n o t a c c u m u l a t ea ny l e gum i ns . T h i s i s a f e a t u r e i n w h i c h t he y r e s e m b l e t he Bromeae/Triticeae. Af u r t he r c o r r e l a t i on o f pa t t e r n s be t w e e n Bromeae~riticeae a n d Festuca/Loliumcon ce rns th e i r f ruc tosan s t ruc tures ; SMrr i-i (1968) f ou nd shor t -ch a in f ruc tosans int he s e t a xa w he r e a s t he o t he r Poeae pr odu c e l ong - c ha i n f r uc to s a ns . C on c e r n i ngpro te in da ta , p ro lam in s imi la r i t i e s r eve a led by BtmcEss & a l. (198 7) a re of in te re s t.T h e s e a u t h o r s d e r i v e d s tr o n g i m m u n o c h e m i c a l a n d m o l e c u l a r a r g u m e n t s i n f av o u ro f c l o s e r e la t i ons h i p s be t w e e n Lolium perenne, Festuca rubra, F. arundinacea, a n dm e m b e r s o f t h e Triticeae ( w he a t , r ye , a nd ba r l e y ) . S e r o l og i c a l l y s e pa r a t e d f r omt he s e s pe c i e s w e r e Dactylis glomerata, Phleum pratense an d Arena sativa w i t hs t rong m u t ua l a n t i ge n i c s i m i l a r it i e s. T h e p r o l a m i n i nve s t i ga t i on o f ES ~ N & H m u( 1989) p r oduc e d t he s a m e r e s u l t : Festuca c lus te r s w i th Hordeum a n d Bromus, n o tw i t h Arena an d Phleum. O n t he c on t r a r y , D N A da t a f a vou r t he " c l a s s i c a l "i nc l u s i on o f t he Lolium gr oup i n t o t he Festuceae ve rsus Bromeae/Triticeae(Soe,ZNG & al. 1990: RF LP ; Hm u & JOHNSON 1991: D N A reasso cia t io n; D Av is &SOm~N6 1993: R FL P; HSIAO & al . 19 95: nu clea r rDN A (ITS) .

CLAYTON & RENVOIZE (198 6) pe rce ive d so m e ge ne ra of the t r ibe Poeaei n c l u d i n g Lolium, Festuca, Vulpia, Castellia, Cynosurus, Lamarckia, a n d Micro-pyrum a s a s e p ar a te p h y l o g e n e t i c li n e ( r o u n d e d l e m m a a n d l i n e a r h i l u m b e i n g t h espec i f i c cha rac te r s ) , bu t they s t r e ss tha t " th ey a re insuf f ic ien t ly d i s t inc t to war ran tf o r m a l r e c ogn i t i on a s s ub t r i be s " . I t i s i n t e r e s t i ng t ha t m os t o f t he ge ne r ae n u m e r a t e d b e l o n g t o o u r a b e r r a n t Lolium group (Table 1 ; Lamarckia not t e s ted ,Cynosurus di f fe rs ) . PRAT (1960) inc lud ed Lolium i n t he Hordeae ( syn . Triticeae),b u t n o t Festuca a n d Vulpia.

S i nc e t he p r o t e i n da t a a r e ve r y d i s t i nc t, t he t a xon om i c pos i t i on a t l e a s t o f t heg e n e r a Festuca, Lolium, Vulpia, Castellia, Lamarckia an d Micropyrum s h o u l d b er e - e va l ua t e d .

Pooideae-Bromeae a n d Pooideae-Triticeae. T he y a r e s i m i l a r i n t he i r p r o t e i np r o f i le s a nd d i f f e r e n t f r om t he o t he r Pooideae. W i t h i n t he p r o l a m i n f r a c t i on , l a rgeM . W . p r o t e i n s a r e c ha r a c t e r i s t i c s ubs t a nc e s . L e gum i ns a r e ne ve r a c c um ul a t e d i ns i gn i f ic a n t a m oun t s , bu t c a n o c c u r oc c a s i ona l l y a s m i no r c om pon e n t s ( e .g . , i nTriticum aestivum: SIN~H & a l. 1991) . T he w e l l kno w n seed pro te in prof i l e s of thece rea l s wh ea t , rye , and ba r ley repre sent the cha rac te r i s t i c pa t t e rn of bo th t r ibes.T he c l e a r s e pa r a t i on o f Bromeae/Triticeae f r om t he r e s t o f t he Pooideae iss uppo r t e d a l s o by o t he r c ha r a c t e r s , e . g . , s i m p l e s t a r c h g r a i n s ve r s us c om poundgr a i n s i n t he o t he r Pooideae tribes (MACFARLANE& WATSON 19 82 , DAHLGREN& al.1985) . A sepa ra t ion o f the Pooideae i n t o t he t w o s upe r t ri be s Poodae a n d Triticodae


12/15

254 L. CHEN & al.:(WATSON a l. 1985, WATSON & DALLWITZ 1992) see m s h igh ly jus t i f ied. F orBrachypodium, our da ta (Table 1 , Triticeae) s ugge s t a n i s o l a t e d pos i t i on ( C A T A L ~& a l . 1995) r a the r than an in tegra t ion in to the Triticeae w h i c h w a s s ug ge s t e d , e. g .,by CLAYTON& RENVOIZE (1 98 6) .

A r u n d i n o i d e a e . R b c L data (BARKER & al . 1 995) ind ica te subfam . Arundino i -deae t o be pa r a phy l e t i c g i v i ng r i s e t o Chloridoideae, Centothecoideae, a n d Pani-coideae. I n ou r s c r e e n i ng t h is s ub f a m i l y i s r e p r e s e n t e d by a f e w s pe c i e s on ly .T he r e f o r e , no s i gn i f i c a n t c onc l u s i ons c a n be p r e s e n t ed . L e gum i ns ( s o l ub l e o runs o l ub l e ) a r e no t a c c um ul a t e d , a nd l ow M . W . p r o l a m i ns ( i n t he r a nge o f 13 -17 kD a a nd 20 - 28 k D a , H I L t: & ESN 1988 ) a r e p r od uc e d a bunda n t ly .

Chloridoideae. T he y s how a p r o t e i n p r o f i l e s i m i l a r t o t he p r o f i l e o f t heArundino ideae , w hi c h c o r r e s ponds w i t h s i m i l a r i t i e s i n m or pho l og i c a l f e a t u r e s(Hm u 1985, CLAYTON 1981). In bo th su bfam i l ie s the low M .W. p ro lam ins ( 2 0 -26 kDa : HILt : & ESEN 1988) con s t i tu te the ty p ica l pro te ins , how ever , ne i th e r the3 8 k D a g l u t e l i n ( e x c e p t Uniola latifolia) nor a ny l e gum i ns oc c u r i n de t e c t a b l ea m oun t s .Panico ideae . T he y a r e c l e a r l y s e pa r a t e d f r om t he o t he r Poaceae s ub f a m i l i e sw i t h a c ons p i c uous a c c um ul a t i on o f g l u t e l i n s w i t h a M . W . o f c a . 38 kD a . T he yobv i ou s l y c ons t i tu t e d i s u l p h i de - l i nk e d d i m e r s o f c a . 21 kD a e a c h . N - t e r m i na la m i no a c i d s e que nc i ng o f a s ubun i t o f t he r e s pe c t i ve Echinochloa crus-gall ipr o t e i n y i e l de d a s e que n c e h i gh l y s i m i l a r t o t he ~ - c o i x i n f r om Coix lacryma-jobia nd ~ - z e i n f r om Zea mays , as rep or te d b y OTTOBONI & a l . (199 3) . W estern blotsu s i ng a Sorghum cernuum 38kD a a n t i s e r um s how e d s e r o l og i c a l c r o s s - r e a c t i v i t yw i t h a l l pa n i c o i d t a xa t e s t e d , bu t f a i l e d f o r a l l t axa f r o m o t he r s ub f a m i l i e s . T hus ,t he s e p r o t e i n s c a n be r e ga r de d a s i m por t a n t d i a gnos t i c p r o t e i n m a r ke r s f o r t hePanicoideae a nd a pp l y t o bo t h m a j o r t r i be s , Paniceae an d Andropogoneae .

S e e d s t o r a g e p r o t e i n s a r e g o o d m a r k e r s f o r a s s e s s i n g t a x o n o m i c a n dphy l oge n e t i c r e l a ti ons h i p s a t va r i ous l e ve l s f r om s pe c i e s t h r oug h s ub f a m i l i e s.T he y a r e p r e s e n t a bunda n t l y a nd t he r e a r e s t a nda r d m e t hods o f e x t r a c t i on a ndana lys i s . D i f fe ren t molecula r s i ze , cha rge and so lubi l i ty , l a rge ly due to thep r e s e nc e o f m u l t i ge ne f a m i l i e s , c a us e s u f f ic i e n t d i f f e re n t i a t ion . T he s c r e e n i ng o fp r o t e i n c ha r a c t er s r e po r t e d he r e a i m s a t t he d i f f e re n t i a t i on o f l a r ge r t a xonom i cgroups . I f a s soc ia ted wi th subfami l ie s , t r ibes and subt r ibes , a spec i f i c pro te inpa t t e r n c a n be e s t a b l i s he d . I nd i v i dua l a c c e s s i ons , s pe c i e s , o r ge ne r a w h i c h s howm a j o r d e v i a t i o n s f r o m n o r m a l p a t t e r n s c a n o f t e n b e a s s i g n e d t o i s o l a t e dphy l oge n e t i c pos i t i ons o r t o i nc o r r e c t t a xon om i c t r ea t m e n t .T hus , s e e d p r o t e i n s a r e e a s y - t o - u s e , w i de l y a pp l i c a b l e m a r ke r s f o r va r i ouss ys t e m a t i c p r ob l e m s .

This investigation has been supported by the Deutsche Forschungsgemeinschaft. Theauthors are also very mu ch ob liged to Dr C. HORSTMAYN,Gatersleben, for sequencing o f the38 kD a protein.

Re f e r enc e sBARKER, N . P., LINDER,H. P., HARLEY,E. H., 19 95: Polyphyly o f Arundinoideae (Poaceae):Evidence f rom rbcL sequence data. - Syst. Bot. 20: 423-435.


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