Biochem. Physiol. Pflanzen 183,153-158 (1988) VEB Gustav Fischer Verlag Jena

Storage Globulins in Barley Grains!)

ERNST WEBER and RENATE MANTEUFFEL Zentralinstitut fur Genetik und Kulturpflanzenforschung

der Akademie der Wissenschaften der DDR, Gatersleben, G.D.R.

• Key Term Index: seed proteins, storage globulins, polypeptide pattern, immunoelectrophoresis, Western blotting, glycoproteins, Hordeum vulgare L.

Summary

Endosperm and embryo tissues were removed by hand dissection from mature caryopses of Hor­deum vulgare L. After sequential extraction of albumins, globulins and prolamins the globulin fraction derived from both tissues was investigated by SDS-polyacrylamide gel electrophoresis and density gradient centrifugation as well as by specific immunological detection of the individual polypeptides and proteins.

Among the seed proteins of most cereal species prolamins constitute the prevailing protein fraction. They have been investigated in many details including the extensive characterization of the corresponding genes (BROWN et al. 1982; KREIS et al. 1985). On the contrary, at present only limited informations are available about the saIt-so­luble globulins of cereal grains, except the globulins from oat caryopses. The latter share close similarities with 78 and 118 globulins of legume seeds (BURGESS et al. 1983; ADELl and ALTOSAAR 1984; ROBERT et al. 1985 a, b). In particular with respect to the 118 globulin it has been demonstrated that these similarities are due to the mode of bio­synthesis on membrane bound polysomes (BRINEGAR and PETERSON 1982; MAT LA­SHEWSKI et al. 1982; ADELl and ALTOSAAR 1983), precursor processing (BRINEGAR and PETERSON 1982; ADELl and ALTOSAAR 1983; ADELl et al. 1984), structural properties (BRINEGAR and PETERSON 1982; BURGESS et al. 1983) and features of the corresponding genes (WALBURG and LARKINS 1986).

In order to characterize the globulins from barley grains we prepared the globulin fractions from endosperm and embryo tissues. They were analyzed by means of bio­chemical and immunological methods.

Mature grains of Hordeum vulgare 1. cv. Salome, were inbibed for 12 h at room temperature. Embryos and endosperms were prepared by hand dissection, immediately frozen in liquid nitrogen and subsequently ground in a mortar under liquid nitrogen. The resulting flour was subjected to sequential extractions of the main seed protein fractions. Albumins were extracted twice using a DMSO-containing acetate-buffered medium before the globulins were removed by two extractions in a phosphate-buffered salt-solution (WEBER et al. 1978; NEL'M _\NN and WEBER 1978; LICHTE N­

FELD et al. 1979). The prolamin fraction was extracted twice with 55 % (v/v) isopropanol/2 % (v/v) 2-mercaptoethanol for 30 min at 60 °C (SHEWRY et al. 1978) . Freeze-dried globulins were used for the immunization of rabbits in order to generate polyspecific antisera (ZUR NIEDEN et al. 1982).

1) The paper was presented at the 4th Symposium on Seed Proteins , held at Gatersleben, G.D.R., July 19-23, 1987.

BPP 183 (1988) 2-3 153

Fig. 1. Fused rocket imnlwlOelectrophoresis of embryo globulins separated on a linear density gradient. Approximately 3 mg globulins including the marker proteins (arrows) vicilin (a) or legumin (b), which were solubilized il\ 0.05 M Tris-maleate, pH 6.9, 1.5 M KC1, were layered onto a 10 ml gradient. Cen­trifugation was performed at 78,000 gav, 2 °C for 18 h. Sedimentation pattern were screened by fused rocket immunoelectrophoresis using anti-embryo globulin antibodies complemented with monospe­cific anti-vicilin or anti-Iegumin-IgG, respectively. The fraction from left to right are from bottom to top of the gradient.

Fig. 2. Crossed immunoelectropherograms of embryo globulins separated by affinity chromatography on Con A-Sepharose 4B. a - non-bound fraction b - bound globulins, eluted with:x-methyl-mannopyrannosid. The globulins were precipitated using anti-embryo globulin antibodies.

Polypeptide patterns were estimated by SDS-polyacrylamide gel electrophoresis (LAEMMLI 1970) after denaturation of the samples as described elsewhere (WEBER and BRANDT 1985). The sedimenta­tion behaviour of globulins was analyzed by ultracentrifugation on a linear sucrose density gradient (7.5-30 %, wjv). The gradient fractions were screened by fused rocket immunoelectrophoresis (AXEL­SEN et al. 1973). Western blotting analyses were performed as described by HELBING and MANTEUF­FEL (1987).

Quantitative estimations revealed that prolamins constitute about 65-70% of the extractable seed protein, whereas albumins and globulins contribute by 15-20% and 10-13%, respectively. Glutelins were not considered in this calculation. Similar amounts of globulins among the seed proteins of barley grains were reported elsewhere (IvANKo 1971, BRANDT 1976). Almost equal amounts of globulins were detected in endo­sperm and embryo samples. Since the embryo, however, contributes only a small por­tion of the total dry matter of the grain there is obviously a relative enrichment of globulins in that part of barley caryopses.

When the embryo globulins were further fractionated by ultracentrifugation on a linear sucrose density gradient, two distinct proteins have been detected by fused rocket

154 BPP 183 (1988) 2-3

Mr'1O-3

80-

60-

40-

20-

2 3

3 4 5 2 3 4

4

8

Fig. 3. SDS-polyacrylamide gel electrophoresis of barley (Hordeum vulgare L.). and field bean (Vicia (aba 1.) globulins on 10 % acrylamide gels followed by staining with Goomassie brillant blue. 1 - fi eld bean vieilin, reduc ed 2 - fi eld bean legumin, non-reduced 3 - field bean legumin, reduced 4 - barley embryo globulins, non-redllced 5 - barley embryo globulins, reduced.

Fig. 4. Western blotting analyses of barley globulins separated by SDS-polyarcylamide gel electropho­resis under reduced and non-reduced conditions. 1- endosperm globulins, reduced 2 - endosperm globulins, non-relluced 3 - embryo globulins, reduced 4 - embryo globulins, non-reduced. After the polypeptides were electrophoretic ally transferred to nitroccllulosem cmbranes the blots were screened with anti-embryo globulin antibodies followed by anti-rabbit-IgG-horse radish per­oxida.se-conjugat. Peroxidase staining was performed by 4-chloro-l-naphthole and H20 2 (HAWKES

et al. 1982).

immunoelectrophoresis of the eluted gradient fractions (Fig. 1). The sedimentation behaviour of these proteins was very similar to the pattern of the 78 globulin viciIin from Vicia {aba seeds. In addition to similar proteins the corresponding fused rocket immunoelectropherograms of oat globulins showed an 118 globulin which was prccipi-

BPP 183 (1988) 2-3 155

tated by anti-oat globulin antibodies. On the other hand, the latter did not precipitate any 11S globulin-like protein from the globulin fractions of barley embryos and endo­sperms (results not shown).

In order to check the barley globulins for a possible glycoprotein nature the glob­ulins were fractionated by means of affinity chromatography on Con A-Sepharose. The sorbent retained the two predominating proteins, which could be released specifically during elution in the presence of lX-methyl-mannopyrannosid (Fig. 2). Thus, our results demonstrate for the first time a glycoprotein nature of barley globulins, which obviously contain mannose-containing oligosaccharide side chains in their constituent polypep­tides. Concerning the well described oat globulins (BURGESS et al.1983; ADELl and ALTO­SAAR 1984) as well as globulins from other cereals there is no indication that the corres­ponding 7S globulins can be considered as glycoproteins, too.

Barley globulins were subjected to SDS-polyacrylamide gel electrophoresis under reducing and non-reducing conditions in order to elucidate the polypeptide patterns. Fig. 3 shows the polypeptide patterns of barley globulins in comparison to those derived from the globulins of Vi cia faba seeds. Obviously, there is no remarkable change in the polypeptide pattern of barley under conditions where disulfide bonds were cleaved. Western blotting analyses using antisera directed against embryo globulins revealed similar results.

Furthermore, the polypeptide pattern of embryo globulins was compared by West­ern blotting with those derived from endosperm samples. The globulins from endo­sperm and embryo tissues were separated on SDS-polyacrylamide gels under reducing and non-reducing conditions followed by Western blotting using antibodies raised against embryo globulins. In agreement with the Coomassie blue-stained electropherograms very similar polypeptide patterns were observed for the globulins from both tissues (Fig. 4). Moreover, the immunological reactivity of the higher molecular weight poly­peptides was affected by the cleavage of disulfide bonds prior to electrophoresis as in­dicated by weaker labelling of bands in the reduced samples.

The globulins of barley and oat have certain antigenic determinants in common. The observed cross-reactivity seems to be attributed to polypeptides of the 7S-like globulins (results not shown). Immunological cross-reactivities between the globulins from different cereals as well as from Pisum seeds have been observed previously by other authors, too (ROBERT et al. 1985 b, c).

At present we do not have any information about the structure of individual glob­ulin components of barley. Nevertheless, it is tempting to conclude, that 7S globulin­like proteins are the prevailing components among the storage globulins of barley grains. They can be considered as glycoproteins.

Acknowledgements

This work was supported by the VVB Saat- und Pflanzgut, Quedlinburg (DDR) and the Akade­mie der Wissenschaften der DDR.

156 EPP 183 (1988) 2-3

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Authors' address: Dr. sc. ERNST WEBER and Dr. se. RENATE MANTEUFFEL, Central Institute of Ge­netics and Crop Plant Research, Academy of Sciences of the G.D.R., CorrensstraBe 3, Gatersleben, DDR - 4325.

Biochem. Physiol. Pflanzen 183, 158 (1988) VEB Gustav Fische~ Verlag J ena

Buchbesprechung

LUGTENBERG, B. (Ed.): Recognition in Microbe-Plant Symbiotic and Pathogenic Inter­actions. Proc. of the NATO Advanced Research Workshop on Recognition in Microbe-Plant Symbiotic and Pathogenic Systems held at Biddinghuizen, The Netherlands, May 11-16, 1986. NATO ASI Series H: Cell Biology, Vol. 4. XIV, 449 Seiten, zahlr. Abb. u. Tab. Springer-Verlag, Berlin-Heidelberg-New York-London-Paris-Tokyo 1986. Preis DM 198.

Erkcnnungsreaktionen zwischen hoheren Pflanzen und symbiontischen oder parasitischen Mi­kroorganismen sind auslosende und regelnde Faktoren cines ausgewogenen Mechanismus, der es den Pflanzen ermoglicht, mit Mikroben in gemeinsamer Umwelt zu bestehen. Die jiinsten Fortschritte bei molckulargenetischen Untcrsuehungen an Rhizobium/Leguminosen- sowie PathogenfWirt-Inter­aktionen forderten zu einer interdisziplinaren Diskussion heraus. Deshalb haben sich Molekular­biologen, Phytopathologcn, l\fikrobiologen, Pflanzenphysiologen, Biochemiker, Bodenbiologen und Agrarwissenschaftler aus 11 Landem in Biddinghuizen versammelt, urn ihre neuesten Forsdmngs­ergebnisse vo]'zutragon sowie Vorochlage fiir kiinftige Untersuchungen zur Aufklarung der moleku­laren Basis von Mikroben/Pflanzen-Intcraktionen zu formulieren. 46 der bei diesem AnlaB von nam­haften Wissenschaftlem aus fiihrenden Instituten dreier Koutinente gehaltenen Vortrage sind unter den Schwerpunkten 1. Erkennung bei der Knollchenbildung durch Rhizobium, 2. Erkennung bei Pathogcn/Pflanzp-Interaktionen, 3. Erkennung bei Abwehrreaktionen, 4. Erkennung bei der bio­logischcn Krankheitsbekiimpfung, is. Empfehlung fiir kiinftige Forschungen und Anwendung im vorliegenden Bueh abgedruckt. Die Palette der darin diskutierten Probleme reicht - urn nur einige zu nennen - von Anheftung, Signalrczeption und -transduktion, Penetration, Elicition und Suppres­sion von Wirtsreaktionen, Genregulation und -expression, Pathogenese bis zum Zelltod. Neben dem hohen wisscnschaftlichen Nivcau und dcr groBen Aktualitat, die jeden Beitrag auszeiehnen, sind die schnelle Drueklegung und Herausgabe des Buches, nur 4 Monate nach der Veranstaltung in sorg­faltig redigierter Form, lob end hervorzuheben. Von der Tagung und dem vorliegenden Bericht werden sieher starke Impulse auf einschlagigc Forschungen und angrenzende Disziplinen ausgehen.

M. SCH~IIEDEKNECHT, Aschersleben

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