P1. Syst. Evol.

206:243-257 (1997)

Plant Systematics and Evolution

Springer-Verlag 1997 Printed in Austria

Accumulation of seed storage proteins and the taxonomy of Poaceae*

LIXUE CHEN, HILDE FISCHER, and UWE JENSEN

Received 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 feature and can be used to additionally characterize plant taxa. To date, mainly crop proteins have been analysed in the Poaceae. In this investigation seed proteins from 147 species were screened with emphasis on legumin-like proteins and prolamins. The groups resulting from evaluation of the protein profiles correspond with well-known subfamilies and tribes. Panicoideae are clearly separated from Pooideae. Within Pooideae, the Bromeae plus Triticeae tribes revealed obvious similarities. Lolium, Festuca and Vulpia, generally included in the tribe Festuceae, revealed a protein profile similar to the profile of the Bromeae/Triticeae. Legumin-like proteins are accumulated abundantly in Bambusoideae and Pooideae except Bromeae/Triticeae, however, only the species included in the Aveninae subtribe produce "soluble"(globulin-type) legumins as already known from Avena sativa.

The Poaceae are one of the largest angiosperm families with ca. 10000 species. They are probably a monophyletic group (DOYLE & al. 1992) and clearly separated from related monocotyledon families by their reduced flowers arranged in spikelets as well as by the starchy endosperm and an embryo with a scutellum-like cotyledon. Together with the Cyperaceae and related monocot families they are characterized by the "wind-pollination syndrome" (DAHLGREN 1983). Several species are ecologically and economically important. Grasslands occupy a third of the land's surface (SCHANTZ 1954), and grasses are the main feeding resources for savannah animals and livestock. Cereal crops (wheat, rye, rice, maize, millet) are essential food components for humans. Although no general agreement has been achieved for the main classification of the family [with a range of only two subfamilies (TZVELEV 1989) up to 13 subfamilies (CARO 1982)], usually the subfamilies Bambusoideae, Pooideae, Centothecoideae, Arundinoideae, Chloridoideae, and Panicoideae are recognized * Dedicated to emer. Univ.-Prof. Dr FRmORICHEHRENDORWRon the occasion of his 70th birthday

244

L. CHEN& al.:

(STEBBINS CRAMPTON1961, CLAYTON& RENVOIZE1986). Within subfamilies, the separation of some tribes and subtribes is still questionable. Within subfam. Pooideae the taxonomic position of the Meliceae and Stipeae is uncertain (DAHLGREN& al. 1985, SOREN6& al. 1990). The Bromeae, interpreted as a separate tribe, e.g., by CLAYTON& RENVOIZE(1986), are included in the "Triticum group" by DAHLGREN & al. (1985), or belong to the tribe Festuceae (PRAT 1960). The relationship of several other taxa is unclear, e.g., of Brachypodium, Catabrosa, Ehrharta, Koeleria, Phippsia, and Uniola (PRAT1960, SORZN6 & al. 1990). CLAYTON& RENVOIZE(1986) discussed several important characters and used them for outlining the grass phylogeny. The tropical Bambusoideae are perceived as the most primitive extant grasses and descendants of the early forest savannah grasses in a semi-open ecotone, which is corroborated by the results from ndhF sequence analysis (CLARK am. 1995). The oryzoid Bambusoideae are perceived as & links to the temperate Pooideae. The other subfamilies, characterized by a mesocotyl, might have a common origin with the Centothecoideae, including Arundinoideae, Chloridoideae, and Panicoideae as independently evolved descendants. Within these three subfamilies the C4 photosynthesis pathway evolved in parallel evolution. Recently DNA data have been successfully used in grass taxonomy and phylogeny. A chloroplast DNA inversion has been detected indicating the monophyly of Poaceae (DOYLE& al. 1992). The subfamily structure outlined by CLAYTON& RENVOIZE(1986) was largely confirmed: In the ndhF sequence analysis (CLARK & al. 1995) the neotropical herbaceous Bambusoideae tribes Anomochloeae and Streptochaeteae appear as sister groups to the rest of the family. Also, a separation of the Poaceae into a "BOP" clade (Bambusoideae plus Oryzoids, and Pooideae) and a "PACC" clade (Panicoideae, Arundinoideae, Chloridoideae, Centothecoideae) has been supported (DAVIS& SORENG1993: RFLP; BARKER& al. 1995: rbcL; CLARK& al. 1995: ndhF). An interesting molecular analysis based on ITS sequences from nuclear rDNA indicated a sister group relationship between Poeae/Aveneae and Bromeae/Triticeae (HslAO & al. 1995), Molecular data also improved the knowledge of certain tribes (e.g., Oryzeae: DUVALL& al. 1993; Poeae: SOR~NG& al. 1990) as well as infrageneric relationships (Bromus: PILLAY& HILu 1995; Festuca: DARBYSHIRE& WARWICK 1992; Hordeum: BAUM & BAILEY 1991; Poa: SORENG1990; Brachypodium: Sra & al. 1993, CATAL~q& al. 1995). Also seed protein characters were used for valuable contributions to the systematics and phylogeny of grasses. The Poaceae seed proteins, being essential nutrients for humans, belong to all four protein groups classified by OSBORNE (1924), i.e. albumins (water soluble), globulins (soluble in dilute salt solutions), prolamins (soluble in aqueous ethanol), and glutelins (soluble in dilute alkali or acid). They have been studied intensively especially in cereals where often prolamins account for >50% of the total endosperm protein (SHOTWELL LARKJNS1989). ~% Wheat (Triticum aestivum and closely related crop taxa), rye (Secale cereale), and barley (Hordeum vulgare) belong to the tribe Triticeae within subfam. Pooideae. Their seed protein profile is dominated by prolamins. They are characterized by an unusual amino acid composition with high proline and amide nitrogen, and low proportions of basic and acidic amino acids. There are three types of prolamins, the HMW, S-poor, and S-rich prolamins, with a high diversity

Seed storage proteins of Poaceae

245

both on the gene and protein level within each group. However, also other proteins are present in smaller amounts, e.g., a legumin-like protein called triticin, detected in Triticum aestivum up to 5% of the total seed protein amount (SINOH& al. 1991). Maize, sorghum and most of the other cultivated millet species belong to subfam. Panicoideae. In seeds of these crop plants prolamins also dominate (called zeins in Zea mays, and kafirins in Sorghum). They are rich in alanine and leucine. In oats (Arena sativa: Pooideae, Aveneae) and rice (Oryza sativa: Bambusoideae, Oryzeae), prolamins are a minor storage component accounting for less than 15% of the total seed nitrogen (SHOTW~LL& al. 1988). In both species legumin-like storage proteins (SHOTWELL & al. 1988, TAKAIWA& al. 1986) are the major component. They are predominantly globulins in Arena, but glutelins in Oryza. Despite their different solubility they are homologous to the classical Fabaceae legumins (ROBERT & al. 1985), a condition which is widespread among angiosperms (own data, unpubl.) and conifers (H~ER & DANK 1996). Grass seed storage proteins have already been investigated systematically by several authors. The prolamin size diversity in Poaceae was studied by Hmv & ESEN (1988) using SDS-PAGE. They found three major subfamily groups, i.e. (1) Bambusoideae/Oryzoideae, (2) Pooideae, and (3) Chloridoideae/Panicoideae/ Arundinoideae, with the Pooideae prolamin pattern as the most variable in terms of molecular weight and subunit heterogeneity. From a serological study conducted by ESEN& HICU(1989), members of Pooideae and Panicoideae showed the greatest dissimilarity, with an intermediate position of Oryza (Oryzoideae), Dendrocalamus (Bambusoideae), and Eleusine (Chloridoideae). Further immunological and electrophoretic studies concerned subfam. Arundinoideae (HILu & ESEN 1990), subfam. Chloridoideae (HILu & ESEN 1993), and the tribes Aristideae (ESEN& HILU 1991) and Triticeae (PELGER1993). The antigenic relationships between barley, rye and wheat have been studied by FELSENSTErN& al. (1984), also confirming a lack of cross-reactivity between prolamins of the two subfamilies Pooideae and Panicoideae. Antisera against crude protein extracts from the Zea mays globulin fraction have been used by KHAVKIN& al. (1979). They distinguished Coix and Sorghum antigens from Zea, Euchlaena, and Tripsacum antigens by serological differences; globulins of the Andropogoneae clearly differed from their counterparts in the Paniceae and were widely apart from globulins in other tribes of subfam. Pooideae. Recently LUTI~ (1991) studied the proteins in several tribes of the Poaceae electrophoretically and immunologically and found that Oryza sativa, Stipa viridula, Agrostis alba, Avena sativa, and Phalaris canariensis produce legumin-like proteins as the major storage protein fraction. In the present investigation we report the screening results for major seed proteins from 147 Poaceae species and 103 genera: (1) The legumin-like proteins with a subunit molecular weight (M.W.) of about 55kDa, characterized by susceptibility to reductive cleavage which results in the formation of two polypeptides of ca. 22 and 40kDa; (2) a prolamin with a M.W. of 38kDa, and (3) the prolamin profile considering especially the high molecular weight proteins. This screening is based on SDS-PAGE. Additionally antisera against Avena sativa legumin and the major seed protein of Sorghum cernuum have been used for crossreactivity experiments. Generally only the accumulation of a protein has been noticed; therefore the production of other proteins in unsignificant amounts cannot

246

L. C~N & al.:

totally be excluded. The results extend our knowledge of the distribution of grass seed proteins beyond the well known cereal proteins. Correlations with defined taxonomic groups contribute towards a better understanding of the intrafamiliar phylogenetic relationships.
Material and methods Plant seeds. About 150 species of 103 genera from 19 tribes have been studied (Table 1). The seed materials were obtained from the following Botanical Gardens, Institutes and Companies: Botanic Garden of the University of Copenhagen, Denmark; Royal Botanic Gardens Kew, England; Jardin Botanique de l'Universit6 de Lirge, Belgium; Jardin Botanique de Bordeaux, France; Andre Kroner, Switzerland; IPK Gatersleben, Germany; Botanischer Garten und Rhododendron-Park Bremen, Germany; Institut ftir Landwirtschaftliche Botanik, Universitfit Bonn, Germany; Borntr~iger GmbH, Offstein, Germany; Fa. Conrad Appel, Darmstadt, Germany; Research Institute for Forestry, Sichuan, VR China. Seeds of several species were collected in nature by Dr PEDROGERSTBER~ER,University of Bayreuth, Germany. Protein extraction and SDS-PAGE. Seeds including the seed coats were ground to powder, extracted in a buffer (1 ml per 100mg seeds) containing 10raM Tris, 80raM glycine, 1 M NaC1, pH 8.5 for 1.5-2h and centrifugated at 12000 g for 15 rain. The supernatant (albumins and globulins) was removed and stored at -20C. The pellet was reextracted twice with the same buffer, followed by three extractions with 70% ethanol to obtain the prolarnin fraction; finally a glutelinic fraction was gained by overnight extraction with a buffer containing 65 mM Tris/HCI, 10% (v/v) glycerol, 2% (w/v) SDS, pH 6.8. All these fractions were analysed by sodium-dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) as described in JENSEN& C~N (1991) using nonreducing and reducing (0.2% (w/v) DTE) conditions. Staining was performed with Coomassie Brilliant Blue according to standard procedures. Preparation of antibodies, immunodiffusion and immunoblotting. The legumin-like protein, the major protein of oat seeds was purified by gel filtration chromatography (AcA Ultrogel 34, IBF) using the globulin extraction buffer. An 38 kDa ~-zein-like glutelin (see Fig. 1) from Sorghum cernuum was separated through SDS-PAGE, followed by electroelution (Biotrap equipment, Schleicher & Schtill). The procedures used for antibody production have been described elsewhere (FISCHER & JENSEN 1992). Ouchterlony imunodiffusion tests were performed essentially according to FISCHER& JENSEN (1992), but under partially denaturing conditions with 4M urea in the gels to keep glutelins in solution. Proteins were transferred from polyacrylamide gels to nitrocellulose membranes according to KIaYsE-ANDERSEN (1984); blocking and subsequent incubations were performed in "Blotto" (0.02 M tris, 0.9% (w/v) NaCI, 5% (w/v) non-fat dry milk) at 40C. Detection of cross-reacting proteins was achieved by the reaction of goat-anti-rabbit horseradish peroxidase (BioRad) with a substrate solution containing 0.025% (w/v) 3,3diaminobenzidinetetrahydrochloride, 0.03% (w/v) COC12, 0.03% (v/v) H202. N-terminal amino acid sequencing. Echinochloa crus-galli proteins (glutelin fraction) were separated by SDS-PAGE under reducing conditions. Transfer of proteins to a PVDF membrane, staining and automated N-terminal amino acid sequencing was done as described in FISCHER& al. (1995). Results

The species examined belong to six genera from four tribes of subfam. Bambusoideae, 65 genera from eight tribes of subfam. Pooideae, three genera

Seed storage proteins of Poaceae

247

from two tribes of subfam. Arundinoideae, eight genera from four tribes of subfam. Chloridoideae and 16 genera of two tribes from subfam. Panicoideae. The results of the electrophoretic analyses are listed in Table 1 and summarized in Table 2. They show that the legumins are accumulated only in Bambusoideae and Pooideae (except in Bromeae/Triticeae and in the Lolium group). Corresponding immunoTable 1. The seed protein profile of Poaceae. A legumins "soluble" (of the globulin-type); B legumins "insoluble" (of the glutelin-type): + + + heavily accumulated, + + accumulated, + present, - not visible in PAGE of crude extracts, ? presence doubtful; C accumulation of a 38 kDa M. W. protein; D prolamins: S only low M. W. proteins (up to 30 kDa); M additionally proteins up to 45 kDa; L additionally high M. W. proteins (M. W. above 45 kDa) present and often accumulated Taxa Subfam. Bambusoideae Bambuseae Bashania fargesii (CAMuS) KENt & Yt Fargesia nitida (M~TF.) KENG Phyllostachys pubescens MAZEL ex LEgAtE Oryzeae Oryza sativa L. Ehrharteae Ehrharta calycina SM. E. longiflora SM. Diarrheneae Diarrhena americana P. B. Subfam. Pooideae Nardeae Nardus stricta L. Stipeae Achnatherum calamagrostis P. B. Milium effusum L. Oryzopsis miliacea (L.) Ascii. & SCHWEtYF. Stipa elegantissima LABmL. S. pennata agg. Poeae Briza media L. Catabrosa aquatica (L.) P. B. Catapodium rigidum (L.) C. E. HuBB. Cynosurus cristatus L. Dactylis glomerata agg. Desmazeria sicula (JACQ.)DUMORT. Echinaria capitata DESF. Lamarckia aurea (L.) MOENCI~ Poa chaixii VmI.. P. compressa L. P. nemoralis agg. t3. palustris L. P. pratensis agg. A B C D

+ + + + + +
m

S S S

+ + +

+ + +

++ +

++ + + +

+ + +

++
m

S
S S S S S

+ + + + + + + + +

+ + + + + +

+ +

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

S S M

M S M M M M M

248 Table 1 (continued) Taxa

Puccinellia distans agg. SesIeria varia agg. Castellia tuberulata TIN. Cutandra maritima BENTH. Festuca arundinacea SCHREB. E gigantea (L.) VILE. E ovina agg. F. pratensis HUBS. E rubra agg. Lolium multiflorum LAM. L. perenne L. L. temulentum L. Micropyrum tenellum L. Vulpia membranacea (L.) LK. V. myuros (L.) GMEL. Hainardeae Parapholis strigosa (DtrM.) C. E. HUBBARD Meliceae Glyceria maxima (HARTM.)HOLMS. Melica ciliata agg. M. nutans agg. Schizachne purpurascens SWALE. Aveneae - Aveninae Aira caryophyllea agg. Arrhenatherum elatius (L.) J. & C. PRESL. Avena barbata POTT ex LK. A. brevis ROTH A. fatua L. A. sativa L. A. strigosa SCHRES. Avenella flexuosa (L.) PARE. Avenochloa albinervis (Bolss.) HOLUB A. pratensis (L.) HOLUB Corynephorus canescens (L.) P. B. Deschampsia cespitosa agg. Gaudinia fragilis P. B. Helictotrichon sempervirens (VILE.) PrLG. Holcus lanatus L. Koeleria glauca (Scrmao.) DC. K. pyramidata agg. Lophochloa cristata (L.) H,a~. Rostraria cristata (L.) TSVELEV Trisetum flavescens agg. Aveneae - Phalaridinae Anthoxanthum odoratum agg. Hierochloe australis (ScriR~.) R. & S c s . Phalaris canariensis L.

L. CHEN al.:

A
m m

B
+ + + + + +

--

--

--

--

m --

--

L L
L

--

--

+ + +

+ + +
D

+ + + + + + + + +

S S

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

++ +

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

++ + + +

M M M M

+ +++
++ ++ -

S
M

+ ++
++ ++

+++ +++
++

+
m

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

S
M S

Seed storage proteins of Poaceae Table 1 (continued) Taxa

Aveneae - Alopecurinae Alopecurus bulbosus GOUAN Ammophila arenaria agg. Apera spica-venti (L.) E B. Beckmannia eruciformis (L.) HosT. B. syzigachne FERNACD Calamagrostis arundinacea (L.) ROTH C. epigeios (L.) ROTH Chaetopogon fasciculatus (Ltc) HAYEK Cinna arundinacea L. Gastridium australe E B. Lagurus ovatus L. Phleum phleoides (L.) KARSTEN Polypogon monspeliensis (L.) DESE Bromeae Boissiera bromoides HOCHST.& S~UD. Bromus inermis LEYS. B. sterilis L. B. tectorum L. Triticeae Aegilops geniculata ROTH A. tauschii Coss. A. ventricosa TAUSCH Agropyron caninum (L.) E B. A. junceum agg. Aneurolepidium sp. Asperella hystrix HUMB. Brachypodium sp. B. pinnatum agg. B. sylvaticum (HuDs.) E B. Dasypyrum sp. Elymus arenarius L. E. dahuricus TURCZ. Eremopyrum sp. Hordelymus europaeus (L.) HAp, Z Hordeum agriocrithon AABERG H. murinum agg. H. vulgare L. Psathyrostachys juncea (FIscH.) NEvs~a Secale cereale L. Taeniatherum crinitum (ScHPaSB.)NEVS~ Triticum aestivum L. T. aethiopicum JAKUBZ. T. dicoccum SCHRAYK. T. durum DESE T. monococcum L. T. spelta L.

249

A
+
-

B
+++
+ + +

C
-

D
S
M

+ + +
-

+++ +++ +++

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

S S S
S S S S S

+
-

+++
+ + + + + +

S
S S

L L L L L L L L L ? ?
S S

L L L L L L L L L L L L L L L L L

250 Table 1 (continued)

Taxa A B

L. Cr~y & al.:

Subfam. Arundinoideae Arundineae Molinia caerulea agg. Notodanthonia buchananii (HooK. f.) Zozov Micraireae Aristida inaequiglumis DOMIN Subfam. Chloridoideae Pappophoreae Enneapogon cenchroides (LIcnz.) HuBs. Eragrostideae- Uniolinae Uniola latifolia Mlco_x. Eragrostideae - Eleusinae Dactyloctenium aegypticum WINED. Eleusine coracana GAERTN. E. tristachya KtrNTH. Eragrostis pilosa (L.) P. B. E. tef (Zucc.) T. ROTTER Eragrostideae - Sporobolinae Sporobolus indicus R. BR. Cynodonteae - Chloridinae Chloris distichophylla LAG. C. truncata R. BR. Cynodon dactylon (L.) PERS. Subfam. Panicoideae Paniceae - Setariinae Brachiaria vilIosa (LAM.) A. CAMUS EchinochIoa 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 - Digitariinae Digitaria sanguinalis ScoP. Paniceae - Cenchrinae Cenchrus tribuloides L. Pennisetum americanum (L.) LEEKE Andropogoneae - Sorghinae Chrysopogon gryllus (L.) T ~ . Sorghum bicolor (L.) MOENCH S. saccharatum (L.) MOENCH Andropogoneae - Andropogoninae Andropogon scoparius Mlcnx. Botriochloa ischaemum (L.) KENG

+
m _ _ - -

S
S

- -

- -

?
- -

S
S

?
S S

+ + + + + + + + + + + + + + + ?

S S S S S S S S S S S S S S S S

Seed storage proteins of Poaceae Table 1 (continued) Taxa

C y m b o p o g o n a m b i g u u s A. CAMUS A n d r o p o g o n e a e - Tripsacinae Tripsacum dactyloides L. Z ea mays L. A n d r o p o g o n e a e - Coicinae Coix lacryma-jobi L.

251

A --

B --

C +
+ + +

D S
S S S

Table 2. Data from the protein screening of Table 1, generalized for the subfamilies of the Accumulation of "soluble" globulinic (glob) and "unsoluble" glutelinic (glut) legumins. B Accumulation of prolamins: S M. W. up to 30 kDa; M M. W. up to 45 kDa; L additionally proteins with M. W. above 45 kDa. C Presence of the 38 kDa protein
Poaceae. A

Taxon
Bambusoideae Pooideae Aveneae: most taxa Aveneae: Aveninae Poeae: L o l i u m group B r o me a e / T r it ic e a e Arundinoideae Chloridoideae Panicoideae

A glut glut glob -

B S S (M) S (M) L
L S S S

C +

diffusion experiments resulted in cross-reactivity of a n t i - A v e n a - l e g u m i n with other P o o i d e a e legumins (no matter whether of the globulin or glutelin type) but not with the B a m b u s o i d e a e legumins. The P o o i d e a e are characterized by prolamins of very different M.W., the production of high M.W. prolamins being restricted to the B r o m e a e / T r i t i c e a e including the L o l i u m group. The 38 kDa glutelin is accumulated exclusively in P a n i c o i d e a e . The respective protein of E c h i n o c h l o a c r u s - g a l l i (21 kDa in its reduced form, see also Fig. 1) was analyzed also by N-terminal amino acid sequencing. This species was chosen for reasons of availability of sufficient plant material. The sequence: V V I P Q C S I A A G A A T I Q Y is strongly similar to the previously reported (OrTOBONI & al. 1993) ~-zeins from maize and their homologues in C o i x and S o r g h u m . The c~-zeins which until now cannot be related to any other known storage protein (SHEWRY 1995) obviously are unique and diagnostic to the tribe P a n i c o i d e a e . This is underlined by our immunoblotting experiments with a n t i - S o r g h u m - z e i n , where cross-reactions occur only with proteins of panicoid species.

252 + 94000 67000 43000 30000 20100 14400 S I a I b ] c I d + + +

L. CHEN& al.:

Is

Fig. 1. SDS-PAGE of Panicoideae seed glutelins using reducing (+) and non-reducing (-) conditions. The 38 kDa protein is indicated by arrows. Observe also its 21 kDa subunits under reduced conditions. S Standard proteins (M.W. in Da), a Setaria glauca; b Panicum miliaceum; c Dig#aria sanguinalis; d Echinochloa frumentacea

Discussion
Bambusoideae. The major seed protein is an "insoluble" (glutelin-type) legumin, with its subunits revealing electrophoretic properties similar to the legumin of the Pooideae. However, missing cross-reactivity with the Avena legumin indicates considerable differences in the molecular structure. Prolamins occur frequently, but they are not a major component. The taxonomic position of Ehrharta is dubious (e.g., P~T 1960). CLAYTON RENVOIZE (1986), CLAkI,: & al. (1995) and others include this genus into the Bambusoideae, or it is believed to be a sister taxon of the Bambusoideae (I~LLOC~ & CAMPBELL1986), or excluded from the Bambusoideae branch at all by SODERSTROM& ELLIS (1987). DAI-ILGREN& al. (1985) link Ehrharta with the Arundinoideae, TZVZLEV (1989) with the Pooideae. Some anatomical similarities also connect Ehrharta with the Aveninae genus Helictotrichon (HIIy 1985). The two Ehrharta species tested by us differ from the other Bambusoideae species in producing globulin-type legumins in considerable amounts (Table 1). This corresponds with subtribe Aveninae within the Pooideae, although missing serological cross-reactions with an antiserum against Avena legumin do not favour a close relationship. Considering all available protein data, the Ehrharta legumin features may be interpreted as an indication of an early branching off of Ehrharta from the bambusoid clade, connected with a change from globulin-like to glutelinlike legumins; but further data are desirable to finally judge the position of this genus. Pooideae. The Pooideae, with the exclusion of the tribes Bromeae and Triticeae, generally accumulate legumin-like proteins and produce particularly small molecular weight prolamins. The legumin-like proteins are in most cases of the glutelin type. Only grasses within the Aveneae-Aveninae produce globulin-type

Seed storage proteins of Poaceae

253

legumins in large amounts. The presence of globulin-type legumins has long been known from Arena sativa (BuRgEss & al. 1983), and is now demonstrated for more or less all members of subtribe Aveninae. An Arena sativa legumin antiserum revealed cross reactivity with the legumins extracted from the Aveninae species, but also with the glutelin-type legumins proteins from other Pooideae. This is explainable by sequence homology of both the globulin-type as well as the glutelin-type legumins, which has been demonstrated for Avena sativa (globulintype) and Oryza sativa (glutelin-type) (ROBERT& al. 1985). The protein data for the genera Festuca, Lolium, and Vulpia, generally included in the Pooideae-Festuceae, do not fit the common characteristics of the Poeae, as they produce prominent high molecular weight prolamins, and do not accumulate any legumins. This is a feature in which they resemble the Bromeae/Triticeae. A further correlation of patterns between Bromeae~riticeae and Festuca/Lolium concerns their fructosan structures; SMrri-i (1968) found short-chain fructosans in these taxa whereas the other Poeae produce long-chain fructosans. Concerning protein data, prolamin similarities revealed by BtmcEss & al. (1987) are of interest. These authors derived strong immunochemical and molecular arguments in favour of close relationships between Lolium perenne, Festuca rubra, F. arundinacea, and members of the Triticeae (wheat, rye, and barley). Serologically separated from these species were Dactylis glomerata, Phleum pratense and Arena sativa with strong mutual antigenic similarities. The prolamin investigation of ES~N & Hmu (1989) produced the same result: Festuca clusters with Hordeum and Bromus, not with Arena and Phleum. On the contrary, DNA data favour the "classical" inclusion of the Lolium group into the Festuceae versus Bromeae/Triticeae (Soe,ZNG & al. 1990: RFLP; Hmu & JOHNSON1991: DNA reassociation; DAvis & SOm~N6 1993: RFLP; HSIAO & al. 1995: nuclear rDNA (ITS). CLAYTON & RENVOIZE (1986) perceived some genera of the tribe Poeae including Lolium, Festuca, Vulpia, Castellia, Cynosurus, Lamarckia, and Micropyrum as a separate phylogenetic line (rounded lemma and linear hilum being the specific characters), but they stress that "they are insufficiently distinct to warrant formal recognition as subtribes". It is interesting that most of the genera enumerated belong to our aberrant Lolium group (Table 1; Lamarckia not tested, Cynosurus differs). PRAT (1960) included Lolium in the Hordeae (syn. Triticeae), but not Festuca and Vulpia. Since the protein data are very distinct, the taxonomic position at least of the genera Festuca, Lolium, Vulpia, Castellia, Lamarckia and Micropyrum should be re-evaluated. Pooideae-Bromeae and Pooideae-Triticeae. They are similar in their protein profiles and different from the other Pooideae. Within the prolamin fraction, large M.W. proteins are characteristic substances. Legumins are never accumulated in significant amounts, but can occur occasionally as minor components (e.g., in Triticum aestivum: SIN~H & al. 1991). The well known seed protein profiles of the cereals wheat, rye, and barley represent the characteristic pattern of both tribes. The clear separation of Bromeae/Triticeae from the rest of the Pooideae is supported also by other characters, e.g., simple starch grains versus compound grains in the other Pooideae tribes (MACFARLANE& WATSON1982, DAHLGREN& al. 1985). A separation of the Pooideae into the two supertribes Poodae and Triticodae

254

L. CHEN& al.:

(WATSON al. 1985, WATSON & DALLWITZ 1992) seems highly justified. For
Brachypodium, our data (Table 1, Triticeae) suggest an isolated position (CATAL~ & al. 1995) rather than an integration into the Triticeae which was suggested, e.g., by CLAYTON& RENVOIZE(1986). Arundinoideae. RbcL data (BARKER& al. 1995) indicate subfam. Arundinoideae to be paraphyletic giving rise to Chloridoideae, Centothecoideae, and Panicoideae. In our screening this subfamily is represented by a few species only. Therefore, no significant conclusions can be presented. Legumins (soluble or unsoluble) are not accumulated, and low M.W. prolamins (in the range of 1317 kDa and 20-28 kDa, HILt: & ESN 1988) are produced abundantly. Chloridoideae. They show a protein profile similar to the profile of the Arundinoideae, which corresponds with similarities in morphological features (Hmu 1985, CLAYTON 1981). In both subfamilies the low M.W. prolamins (2026 kDa: HILt: & ESEN 1988) constitute the typical proteins, however, neither the 38kDa glutelin (except Uniola latifolia) nor any legumins occur in detectable amounts. Panicoideae. They are clearly separated from the other Poaceae subfamilies with a conspicuous accumulation of glutelins with a M.W. of ca. 38 kDa. They obviously constitute disulphide-linked dimers of ca. 21 kDa each. N-terminal amino acid sequencing of a subunit of the respective Echinochloa crus-galli protein yielded a sequence highly similar to the ~-coixin from Coix lacryma-jobi and ~-zein from Zea mays, as reported by OTTOBONI& al. (1993). Western blots using a Sorghum cernuum 38kDa antiserum showed serological cross-reactivity with all panicoid taxa tested, but failed for all taxa from other subfamilies. Thus, these proteins can be regarded as important diagnostic protein markers for the Panicoideae and apply to both major tribes, Paniceae and Andropogoneae. Seed storage proteins are good markers for assessing taxonomic and phylogenetic relationships at various levels from species through subfamilies. They are present abundantly and there are standard methods of extraction and analysis. Different molecular size, charge and solubility, largely due to the presence of multigene families, cause sufficient differentiation. The screening of protein characters reported here aims at the differentiation of larger taxonomic groups. If associated with subfamilies, tribes and subtribes, a specific protein pattern can be established. Individual accessions, species, or genera which show major deviations from normal patterns can often be assigned to isolated phylogenetic positions or to incorrect taxonomic treatment. Thus, seed proteins are easy-to-use, widely applicable markers for various systematic problems.
This investigation has been supported by the Deutsche Forschungsgemeinschaft. The authors are also very much obliged to Dr C. HORSTMAYN, Gatersleben, for sequencing of the 38 kDa protein.

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