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IMMUNOLOGICAL SCREENING for SPECIFIC PROTEIN CONTENT in BARLEY SEEDS by ULLA RASMUSSEN

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IMMUNOLOGICAL SCREENING for SPECIFIC PROTEIN CONTENT in BARLEY SEEDS by ULLA RASMUSSEN Carlsberg Res. Commun. Vol. 50, p. 83-93, 1985 IMMUNOLOGICAL SCREENING FOR SPECIFIC PROTEIN CONTENT IN BARLEY SEEDS by ULLA RASMUSSEN Department of Biotechnology, Carlsberg Research Laboratory, Gamle Cadsberg Vej 10, DK-2500 Copenhagen Valby and Department of Physiology, Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500 Copenhagen Valby and Institute of Genetics, University of Copenhagen, Oster Farimagsgade 2A, DK-1353 Copenhagen K. Keywords: Lysine, chymotrypsin inhibitor 2, fluorescent labelling, fixation, mutants A screening method employing immunofluorescence has been developed to judge the amount of specific polypeptides, such as the high lysine protein chymotrypsin inhibitor 2 (CI-2) in the endosperm of barley. Thin sections or sanded seeds were prepared fixed in 4% paraformaldehyde and labelled with antibody against C1-2 conjugated with fluorescein isothiocyanate. With this technique single seeds can be evaluated without destroying their viability. The deposition of CI-2 was followed during the grain development period in seeds of Hiproly and of the Hiproly sisterline (C14362) both by the immunofluorescence technique and by rocket immunoelectrophoresis. Accumu- lation of CI-2 started about eight days later in CI 4362 than in Hiproly. The demonstrable amounts of CI-2 in CI 4362 were 20-25% of the amounts found in Hiproly. 1. INTRODUCTION The discovery of increased lysine levels in The seeds of cereal plants constitute a major mutants in maize, originally selected by their source of~nergy and protein in the diet of man altered opaque kernel phenotypes (37, 38, 43), and domestic animals. The low content of essen- gave an impetus to search for improved protein tial amino acids such as lysine in the major quality in other cereals. By using the dye binding storage proteins of cereal seeds, e.g. the hordeins capacity analysis for basic amino acids com- in barley, limits their nutritional value. Theore- bined with nitrogen analysis, at least eighteen tically protein quality could be improved by mutants in barley have been identified in the changing the relative composition of the seed past 15 years (8, 50). In sorghum the same proteins either by decreasing those poor in lysine techniques revealed that two opaque mutants or by increasing those rich in lysine. had increased lysine contents (49). In all investi- Abbreviations: BSA = bovine serum albumin; CI- 1 = chymotrypsin inhibitor 1; CI-2 = chymotrypsin inhibitor 2; FITC = fluorescein isothiocyanate; IgG = immunoglobulin G; PBS = phosphate buffer saline; SDS-PAGE = sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Springer-Verlag 0105-1938/85/0050/0083/$02.20 U. RASMUSSEN: Immunological screening for barley proteins gated mutants, the lysine increase is accom- diffusion test (30). A description of Hiproly and panied by 10-50% decreases in yield, which is not CI 4362 can be found in (40, 41). surprising given the insensitive nature of the For analysis of CI-2 deposition during the screening technique employed. grain filling period, spikes were collected from In all cases studied, the major effects of the Hiproly and CI 4362 plants grown at Hyldager- mutations have been confirmed by molecular gard in 1984. At 14, 18, 22, 26, 30 and 44 days studies at the polypeptide level. In barley the after anthesis ten spikes were harvested, frozen mutation hor-2ca (mutant Riso 56) specifically in liquid N2 and stored at -20 ~ eliminates the 6-8 unique B hordein polypep- tides (9, 46), the mutation lys3a (mutant Riso 1508) decreases synthesis of all hordein polypep- 2.2. Chemicals tides coded for by the three genes Hot-l, Hor-2 Sephadex G-25 and Protein-A coupled Sepha- and Hot-3 (4), while the mutation Iysl (identi- rose were from Pharmacia Fine Chemicals, fied in Hiproly) increases synthesis of four albu- Uppsala, Sweden. Diethylaminoethyl (DEAE)- min proteins, namely chymotrypsin inhibitor 1 cellulose type DE-52 was from Whatman, and 2 (CI- 1, CI-2), protein-Z and p-amylase (22). Maidstone, England. Bovine serum albumin In maize the opaque-2 mutation seems analo- (BSA) was obtained from Sigma Chemical, St. gous to hor-2ca in selectively decreasing the 22.5 Louis, Missouri, USA. Fluorescein isothiocya- kD zein class and the opaque-7 and floury-2 nate (FITC) was from Research Organic, Cleve- mutations to lys3a in generally inhibiting syn- land, Ohio, USA. Rabbit antimouse immuno- thesis of all zein classes (5). globulin, IgG (control serum) was obtained from To find a commercially useful high lysine DAKO, Copenhagen, Denmark. Para- cereal, a screening technique capable of detect- formaldehyde analytical grade was from Merck, ing changes in individual polypeptides is desira- Darmstad, Germany. ble. Today this should be feasible for any pu- rified protein against which antibodies can be produced. By conjugating a fluorochrome to the 2.3. Fixation and sectioning of tissue antibody and then exposing the appropriate seed Half seeds sectioned by a dorsoventral longi- surface to the fluorescent antibody, the amount tudinal cut were fixed by imbibition in the of the specified protein should be quantifiable. paraformaldehyde-lysine-periodate fixative de- Herein the potential of the immunofluorescent tailed by MCLEAN (36) as described by GIBBONS approach to screen for new mutants is demon- (14). strated for the lysine rich CI-2 protein in the Seeds were embedded in Tissue Tek II, em- endosperm of barley. This technique is far more bedding medium for frozen tissue specimens, rapid than techniques employed hitherto and (Miles Laboratories, Naperville, Illinois, USA) has the additional advantage of not destroying and 12 ktm sections were cut on a Reichert Jung the seed. Frigocul 2700 Cryotome (Jung, Heidelberg, Germany). The sections were transferred to moist 1% gelatine-coated slides. For surface fixation the seeds were embedded 2. MATERIALS AND METHODS in a Cernit-plate (23), and sanded obliquely with 2.1. Plant material a sanding-machine without damaging the em- Seeds of Hiproly barley were from plants bryos. A 4% solution of paraformaldehyde was grown under standard conditions (10) in the applied to the sanded surface of the seed with a Phytotron at the Swedish University of Agricul- paint brush. After two hours at 5 ~ the seeds tural Science, Stockholm in 1983. Seeds of the were washed for 30 min by immersion in phos- Hiproly sisterline, CI 4362, were from plants phate buffer saline, PBS (10 raM-sodium phos- grown at the Carlsberg Research Farm, Hylda- phate containing 150 mM-sodium chloride at gerg~lrd, in the summer of 1981. The genotypes pH 7.3). of the seeds were confirmed by a radial immuno- 84 Carlsberg Res. Commun. Vol. 50, p. 83-93, 1985 U. RASMUSSEN: Immunological screening for barley proteins 2.4. Rocket immunoelectrophoresis of antibody the solution was removed after 15 The extraction of CI-2 was performed as min, and reapplied and the section left for a described by JONASSEN (30). Five ~tl of each further 15 min. Following removal of the BSA extract was placed in a well of a 1% agarose gel. solution, 30 gl FITC coupled rabbit anti CI-2 The electrophoresis buffer was 73 mM-Tris, 24.3 (0.7 rag- ml") or control serum was applied and mM-barbital, 0.34 mM-calcium lactate, pH 8.6. the incubation procedure repeated. The sections The agarose gel contained 16 I,tl CI-2 rabbit were washed by dipping in distilled water and antiserum per ml gel. Rocket immuno electro- then immersing in PBS for 15 min. They were phoresis was run at 2.5 V/cm for 18 hours, and then mounted in glycerol, adjusted to pH 7.4 the protein precipitations visualized by staining with sodium phosphate buffer, stored in the dark with Coomassie blue R-250. at 4 ~ and analysed within 24 hours. 2.5. Test of serum and purification of IgG 2.7.2. Immunolabelling on surface fixed seeds Rabbit antiserum, prepared against purified The surface of the Cernit block containing the CI-2 (28) was obtained from DAKO. The antise- partly embedded and sanded seeds was covered rum was previously tested for monospecificity with PBS for 30 min. The inverted block was by crossed immunoelectrophoresis with a 5 placed in a 6 ml box completely filled with 10% times concentrated crude Hiproly extract (28) BSA in PBS, and the exposed seed surface and by an immune-blotting assay (25) imploy- washed by mechanical shaking for 30 min. The ing purified CI-2 and crude Hiproly extract. A same procedure was then used to incubate with single band at identical positions was obtained in FITC labelled anti CI-2 (0.7 mg ml 4) for 20 min both cases. Purification was accomplished by and then to wash four times with PBS. The block Protein-A Sepharose affinity chromatography was stored at 4 ~ in the dark for one hour before according to the manufacturer's directions. analysis. 2.6. Conjugation of FITC to purified lgG 2.8. Fluorescence microscopy The conjugation between FITC and IgG was A Reichert Univar microscope (Reichert, performed according to (33). The FITC-IgG Vienna, Austria), fitted with a 200 W DC mer- conjugate was separated from free FITC by gel cury lamp (Illumination Industries Inc., Sunny- filtration on a Sephadex G-25 column. The vale, California) was used. Excitation filters, conjugate was then chromatographed on a dichroic mirrors and emission filters were from DEAE-cellulose column to remove under and Optisk Laboratorium (Lundtofte, Denmark) over conjugated IgG. The absorbances at 280 and had the following characteristics: Excitation nm and 495 nm were recorded on a spectropho- filters had a band pass BP 450-495 nm (used for tometer (Shimadzu, UV-120-12, Japan). The the illustrations in Figures 3, 4, 5 and 6) or BP protein concentration was determined (54) and 420-500 nm (used for Figure 1).
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