Electrophoretic and immunological detection of legumin-like seed proteins in several monocot species including several tribes of the Poaceae

Plant Science, 78 (1991) 185--192 Elsevier Scientific Publishers Ireland Ltd.

185

Electrophoretic and immunological detection of legumin-like seed proteins in several monocot species including several tribes of the Poaceae Dawn S. Luthe Department t~]"Biochemistry and Molecular Biology. Mississippi State UniversiO'. Mississippi State. M S 39762 (U.S.A.) {Received February 4th, 1991: revision received May 28th, 1991: accepted May 29th, 1991}

SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis were used to determine the prevalence of leguminlike proteins in seeds from plants representing nine orders of Monocotyledoneae including sixteen tribes of the Poaceae. SDS-PAGE indicated that most monocotyledonous plants examined had major storage protein fractions that could be classified as the I1 S legumin-like globulins. SDS-PAGE and Western blot analysis indicated that representatives of five tribes of the Poaceae {Graminales), A vena sativa L., Or.v:a sativa L., Agrostis alba L., Stipa viridula Trin. and Phalaris eanariensis L. also contained legumin-like proteins as their major storage protein fraction. Key wards: storage proteins: legumin: monocotyledons; Graminales; Poaceae

Introduction

Seed storage proteins have been classically categorized according to their solubility in water (albumins), salt solutions (globulins), alcohol (prolamines) and dilute alkali or acid (glutelins) [1]. In many dicotyledonous plants the most abundant of these fractions is the globulin fraction which is typically composed of the vicilin-like and ieguminlike proteins [2,3]. The legumin-like proteins have sedimentation coefficients in the range of 11-12 S, are comprised of six acidic (or) and six basic (/3) subunits joined by disulfide bonds, and have an apparent molecular weight of about 360 kDa. The a-peptides have acidic isoelectric points and range in size from approximately 30-40 kDa and the/3peptides have basic acidic isoelectric points and apparent molecular weights of about 20 kDa [2]. The t~ and/3 peptides are synthesized as a preproCorrespondence to: D.S. Luthe, Department of Biochemistry and Molecular Biology. P.O. Drawer BB, Mississippi State, MS 39762. U.S.A.

tein with an apparent molecular weight of approximately 60 kDa. The preprotein contains a signal sequence required for movement into protein bodies and a tandem arrangement of the ct and/3 subunits connected by several amino acids [3]. Following translation the precursor is cleaved resulting in a dipeptide comprised of an c~ subunit and/3 subunit joined by a disulfide bond [3]. Six of these dipeptides assemble to form the 11-12 S holoprotein [3]. The primary storage protein fraction in most cereals is the prolamine fraction. However, two of the cereals, oats (Arena sativa L.) [4,5] and rice (Ory-a sativa L.) [6], are known to have leguminlike proteins as a major seed protein component. The major rice storage protein has solubility properties of glutelin, but has biophysical properties similar to other 11 S seed proteins [6]. DNA sequence analysis indicates that the amino acid homology between oat globulin and rice glutelin is about 72% [7,8]. The amino acid homology of either oat globulin or rice glutelin with soybean glycinin or pea iegumin is approximately 35'¼,

0168-9452/91/$03.50 © 1991 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

186 [7,9]. This homology indicates that rice glutelin and soybean glycinin may have evolved from a common ancestral gene [9]. Work by Boroto and Dure [10] suggests that all globulin fractions were derived from two ancestral genes: one for vicilinlike proteins and one for legumin-like proteins. Immunological studies also indicate partial homology among the various seed protein classes. Millerd [11] has shown that various legumin-like proteins among the Fabaceae (Leguminales) are immunologically related. Alexenko et al. [12] determined that legumin-like proteins from several dicots were immunologically related to the soybean legumin-like protein, glycinin. Several groups have shown immunological similarity among oat globulin, rice glutelin and pea (Pisum sativum L.) or soybean (Glycine max L.) legumin [5,13,14]. Also, Western blot analysis indicated that antibody raised against oat globulin [15] or the o~subunit of rice glutelin [14] cross-reacted with several polypeptides from wheat seeds. The rice antibody reacted with a group of wheat (Tr#icum aestivum L.) polypeptides in the molecular weight range between 43 and 68 kDa; however, it did not cross-react with seed protein extracts from other major cereals, including barley (Hordeum vul,~,are L.), rye (Secale cereale L.), sorghum (Sorghum hicolor Moench.), and maize (Zea mays L.) [14]. Various other studies have investigated the relationships among the cereal prolamines [3,16,17]. The purpose of this work was to use electrophoresis (SDS-PAGE) and immunoblot analysis (Western blot) to determine the prevalence of lequmin-like proteins in representatives of the Monocotyledoneae and the tribes of the Poaceae (Graminales). Materials and Methods

Plant material Some of the seeds were collected locally in Starkville, MS. Dr. James Delouche, Seed Technology Laboratory, and Dr. Sidney McDaniel, Department of Biological Sciences, Mississippi State University, donated seeds from the Poaceae and from several other monocotyledon families, respectively. Other seeds were obtained from Thompson and Morgan Seed Company (Jackson, N J).

Protein preparation and electrophoresis Seeds were ground to a fine powder using a mortar and pestle. Protein was extracted by homogenizing the powder in sodium dodecyl sulfate (SDS) sample buffer [18] containing I mM phenyl-methyl-sulfonyl fluoride (PMSF) and 5% (v/v) /3-mercaptoethanol. After homogenization the extract was boiled for 5 min, cooled to room temperature and centrifuged at 13 000 × g for 5 min. The pellet was discarded and the supernatant fraction was analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a 10-15% (w/v) linear gradient of polyacrylamide. If the gels were not analyzed by immunoblot techniques they were stained with Coomassie brilliant blue. Antibody preparation Rabbit polyclonal antibodies were prepared against oat globulin and the acidic subunit of rice glutelin as previously described [19]. Rabbit polyclonal antibodies against the 22 kDa subunit of zein, rice prolamine and soybean glycinin were provided by Dr. Jan Miernyk, Dr. Tom Okita and Dr. Niels Nielson, respectively. Western hlot analysis Following separation by SDS-PAGE the proteins were blotted onto nitrocellulose [20] using the ABN polyblot system (American Bionetics, Inc., Hayward, CA) following the manufacturer's directions. Western analysis was performed using the method described by Turner [21]. lmmunoreactive proteins were detected by incubating the membranes in the presence of alkaline phosphatase-conj ugated goat-anti-rabbit immunoglobulin G (Sigma Chemical Co., St. Louis, MO). Naphthol AS-BI phosphoric acid and Fast Blue RR salt (Sigma Chemical Co., St. Louis, MO) were used as substrates for the alkaline phosphatase, immunoreactive peptides were visualized as blue bands following the alkaline phosphatase reaction. Results

Because a survey of representatives of 58 orders of Dicotyledoneae indicated that legumin-like proteins were the predominant storage protein class

187 Table I. Representatives of Monocotyledoneae analyzed by SDS-PAGE. Family

Genus species

Common name

Monocotyledons Butomales Alismatales Juncales Commelinales Zingerberales Liliales Amaryllidales Iridales Palmales Palmales

Butomus umhelhttus L. Alisma suheordatum RaE Juneus tenuis Willd. Commelina eoefistis Willd. Amomum suhuhttum Rob. A,waragus qffh'inalis L. Allium cepa L. lris/oeti~Dssima L. Beaurearnea reeurivata Lem. Sahal minor Pers.

Flowering rush Water plantain Rush Dayflower Cardamon Garden asparagus Onion Iris Ponytail pahn Palm

(data n o t s h o w n ) , a n a l y s e s were d o n e to d e t e r m i n e if this was also the case for several o r d e r s w i t h i n the M o n o c o t y l e d o n e a e . T a b l e I lists the m o n o c o t species tested, a n d Fig. 1 s h o w s S D S - P A G E a n a l y sis o f the seed p r o t e i n s . A l t h o u g h the p r o t e i n c o n c e n t r a t i o n was low for B u t o m u s u m b e l l a t u s L . (lane 1) a n d A l i s m a s u b c o r d a t a Raf. (lane 2), they a p p e a r e d to have very small a m o u n t s o f the

Lanc numbcr IFig. 1) I

2 3 4 5 6 7 8 9 I0

l e g u m i n - l i k e p r o t e i n s . All o t h e r m o n o c o t r e p r e s e n tatives tested h a d p r o t e i n b a n d s that m i g r a t e d in the 3 0 - 4 0 k D a a n d 20 k D a range. T h e s e l e g u m i n like storage p r o t e i n f r a c t i o n s were p r o m i n e n t in all orders, except the P a l m a c e a e (lanes 9 a n d 10). Past studies [3] have i n d i c a t e d t h a t o n e o f the largest o r d e r s w i t h i n the m o n o c o t s , the Poaceae, store m o s t o f their seed p r o t e i n s as the a l c o h o l - s o l u b l e

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MONOCOTS Fig. I. SDS-PAGE analysis of seed extracts from representatives of Monocotyledoneae. Sec Table 1 for the species and hme number designations. Numbers on the right refer to the molecular weight in kDa of the marker proteins (lane M). Numbers on the left indicate the molecular weight (kDa) for some of the major storage protein bands. Open arrows indicate typical legumin-like proteins. Species with legumin-like proteins as a major storage protein class are marked with a star f ~ .

188

prolamine fraction, and not as the salt-soluble globulin fraction. According to Hutchinson [22] the Poaceae were derived from the Juncales. Although only one species, Juncus tenius Willd, from this large order was tested (Fig. 1, lane 3), it had major protein bands in the range of 30-40 kDa and 20 kDa characteristic of legumin-like proteins. Members of two Poaceae tribes, A vena sativa L. and Oryza sativa L., have legumin-like proteins instead of prolamines as a major storage protein class. These proteins have o~- and fl-subunits that are similar to those found in dicots [4,6]. To determine if other tribes within the Poaceae contained the legumin-like seed proteins, representatives from several tribes (Table II) were collected and their seed proteins were analyzed by SDS-PAGE (Fig. 2). Most of the seeds did not contain proteins that could easily be recognized as the c~- and flsubunits of the iegumin-like globulins. However, several tribes did have stainable amounts of proteins similar to the legumin-like globulins. In addition to Arena sativa L. (lane 8) and Oryza sativa L. (lane 13), these were Agrostis alba L. (lane 9), Stipa viridula Trin. (lane 10) and Phalaris canariensis L. (lane 12). Western blot analysis was used to further in-

Table II.

vestigate the relationships among the proteins in the Poaceae. Antibody raised against the 11 S globulin of Avena sativa L. [19] was used to probe blots of the proteins from Fig. 2. Figure 3 indicates that the oat antibody recognized proteins in the appropriate molecular weight range from Agrostis alba L. (lane 9), Stipa viridula Trin. (lane 10), Phalaris canariensis L. (lane 12) and Oryza sativa L. (lane 13). But in addition to these species the oat antibody reacted with some of the proteins from Festuca elatior L. (lane 1), Triticum aestivum L. (lane 2) and Cortaderia selloana (Shult.), Aschers. and Graebn. (lane 4). Two groups of Festuca elatior L. proteins with apparent molecular weights of approximately 66 kDa and 35 kDa cross-reacted with the oat antibody. There was a cross-reactivity between the oat globulin antibody and a group of Triticum aestivum L. proteins with apparent molecular weights between 28 kDa and 49 kDa. In addition, two protein bands of 49 kDa and 28 kDa from Cortaderia selloana (Shult.), Aschers. and Graebn. were recognized by the oat antibody. None of these proteins have molecular weights or electrophoretic patterns typical of the legumin-like globulins, yet they appear to be immunologically related. When a similar blot (Fig. 4) was probed with an-

Representatives of the Poaceae analyzed by SDS-PAGE and Western immunoblot.

Tribe

Genus species

Common name

Festuceae Hordeae Pappohoreae Arundineae

Festuca elatior L. Triticum aestivum L. Pappophorum hicolor Fourn. Cortaderia selloana (Schult.). Aschers. & Graebn. Eragrostis curvuhl (Schrad.) Nees. Sporoholus co'ptamlrus Torr. Cynodon dactylon L. Arena sativa L. Agrostis a/ha L. Stipa viriduht Trin. Zoysiu japonica Steud. Phaktris canariensis L. Oryza sativa L. Panicum mi/iaceum L. Sorghum hicolor Moench. Zea mays L.

Meadow rescue Wheat

Eragrosteae Sporoboleae Chlorideae Aveneae Agrosteae Stipeae Zoysieae Phalarideae Oryzeae Paniceae Andropogoneae Maydeae

Pampas grass Weeping love grass Drop seed Bermuda grass Oat Red top Green needle grass Zoysia Canary grass Rice Proso millet Sorghum Corn

Lane number (Fig. 2) I 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

189

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POACEAE Fig. Z SDS-PAGE analysis of seed extracts from representatives of tribes from the Poaceae. See Table II for the species and lane number designations. Numbers on the left refer to the molecular weight in kDa of the marker proteins (lane M). Numbers on the right indicate molecular weight (kDa) for some of the major storage protein bands. Open arrows indicate typical legumin-like proteins. Species with legumin-like proteins as a major storage protein class are marked with a star ('Jr).

tibody raised against the ~t-subunit of rice glutelin there was cross-reactivity between the antibody and the ct-subunits of Avenae sativa L. (lane 8), Agrostis alba L. (lane 9), Stipa viridula Trin. (lane 10) and Phalaris canariensis L. (lane 12). The antibody recognized the 66 kDa band of Festuca elatior L. (lane l), but not the lower molecular weight band which was recognized by the oat antibody raised against both the ct- and ~-subunits of oat globulin. This indicates that the lower molecular weight band of Festuca elatior L. may be similar to the/~-subunit of the oat globulin. The rice antibody recognized only the 28 kDa band of Cortaderia selloana (Schult.), Aschers. and Graebn. (lane 4) indicating that the 49 kDa immunoreactive peptide was homologous to the /3subunit of oat globulin. The rice antibody reacted with the same wheat (Triticum aestivum L., lane 2)

proteins as the oat antibody suggesting that these proteins are immunologically similar to the c~subunits of both oat globulin and rice glutelin. Others [14,15] have also shown that antibody raised against oat globulin and the a-subunit of rice glutelin reacted with wheat proteins in a similar size range. In addition, both groups [13,14] determined that oat globulin and rice glutelin were immunologically cross-reactive. When a similar blot was probed with rabbit polyclonai antibody raised against rice prolamine, there was no cross-hybridization with most of the species on the blot (data not shown). This confirms work of Okita et al. [14] who have reported that this antibody did not cross-react with prolamines of oat, rye, wheat, barley, sorghum and maize. The only cross-hybridization that occurred was with proteins migrating in the 24 kDa range of Agrostis

190

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Fig. 3. Western blot analysis of a gel similar to that in Fig. 2. The blot was probed with rabbit polyclonal antibody raised against oat globulin. Numbers on right and left indicate thc molecular weight (kDa) of major inamunoreactive polypeptides.

alba L. (data not shown). This is the same molecular weight range as the avenins (prolamines) from oat [24]. When blots of the monocot proteins were probed with anti-glycinin (soybean legumin-like protein) and anti-oat globulin antibody, no cross-reactivity was observed. When the monocot blot was probed with anti-zein (antibody raised against the major prolamine fraction from Zea mays L.) several immunoreactive peptides were found in seed protein extracts from Iris foetidissima L., but with no other monocot species. In contrast to the results of others [13,14] we did not observe cross-reactivity when blots of the grass seed proteins were probed with anti-glycinin antibody. However, there was strong cross-reactivity between anti-zein antibody and proteins from Sorghum bicolor Moench. and Panicum miliaceum

L. (data not shown), two grasses that are closely related to maize. Discussion

Although it is known that legumin-like proteins are present in the seeds of many dicotyledons, there is little information about their prevalence in monocotyledon seeds other than the cereals (Poaceae). This study indicated that the major storage protein class in several monocotyledon species were proteins that had iegumin-like characteristics when analyzed by SDS-PAGE. However, these proteins did not cross-react with anti-glycinin and anti-oat globulin antibody suggesting that the proteins may have limited homology to the soybean and oat proteins. To extend this study representatives of the

191

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Fig. 4. Western blot analysis of a gel similar to that in Fig. 2. The blot was probed with rabbit polyclonal antibody raised against the ot-subunit of rice glutelin. Numbers on the right and left indicate molecular weight (kDa) of major immunoreactive peptides.

Poaceae tribes were analyzed. In addition to oat and rice, three other representatives of the Poacea, Agrostis alba L., Stipa viridula Trin. and Phalaris canariensis L. also accumulate legumin-like proteins as a major storage protein class. This is unusual because most members of this order accumulate the alcohol-soluble prolamines as their principle storage proteins [23]. In oats, prolamine and globulin fractions, respectively comprise about 10% and 80% of the total seed protein [23]. A recent study of oat storage protein genes has shown that there were about 25 avenin genes and 50 globulin genes per haploid genome [25]. However, quantification of m R N A abundance during seed development indicated that there are nearly equivalent amounts of avenin and globulin mRNAs [251. This suggests that some type of posttranscriptional or translational control regulates

the relative abundance of prolamines and globulins in the oat seed. Perhaps some type of regulatory mutation occurred during evolution of the grasses that accounts for the differential accumulation of these two classes of seed proteins in the Poaceae. Since legumin-like proteins were prevalent in most of the monocot seeds tested, it would be interesting to know when and how the shift from legumin-like protein to prolamines occurred in the Poaceae.

Acknowledgements ! would like to acknowledge the fine technical assistance of Ms. Sharon Jeanson, who prepared the seed extracts and conducted the electrophoretic analysis. This work was supported by the Mississippi Agricultural and Forestry Experiment Station, publication no. J-7512.

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