On the localisation and transport of α-amylase during germination and early seedling growth of Hordeum vulgare

0 Department of Biotechnology, Carlsberg Research Center Gamle Carlsberg Vej 10 , DK-2500 Copenhagen Valby by A procedure for the isolation of a germination-specific a-amylase isoenzyme from Hordeum vulgate (cv. Nordal) is described. The pure a-amylase isoenzyme was used to obtain a monospecific anti-a-amylase antibody. The antibody was subsequently used to localise a-amylase in sections of germinating barley seeds (cv. Nordal). Immunohistochemical determination of the pattern of enzyme movement during germination and early seedling growth revealed that at the commencement of germination the a-amylase protein moiety moves away from the entire face of the scutellum and intermediate crushed cell layer. As early seedling growth continues, the protein begins to be synthesised in the aleurone layer and transported away from this layer into the endosperm. - enzyme purification, affinity chromatography, tissue fixation 1. I N T R O D U C T I O N The localisation and transport of hydrolytic enzymes in seeds of germinating barley (Hordeum vulgare L.) have both been widely investigated. Although as early as 1886 (21) certain theories were available to explain the biochemical changes occurring in germinating seeds of the Gramine~e, the processes have as yet not been fully explained. Certain fundamental questions, such as the role of the scutetlum contra the role of the aleurone in the production of hydrolases during germination and early seedling growth, are still under discussion (18). The advent of immunohistochemical techniques suitable for use with plant tissues has allowed reexamination of the localisation and transport mechanisms in vivo rather than in isolated tissues, tissue extracts, or seeds with defined physical damage. The experiments reported in the present work were designed to investigate the localisation and transport of one of the major hydrolytic enzymes produced during germination - the 1,4-a-Dglucan h y d r o l a s e - a-amylase (E C 3.2.1.1.) in germinating seeds of Hordeum vulgare L. cv. Nordal. Nordal was chosen as it is one of the major malting barleys in Denmark. 2. MATERIALS AND M E T H O D S 2.1. Chemicals Agarose type HSB was obtained from Litex, Glostrup, Denmark. Epoxy-activated sepharose 6B and Phadebas a-amylase test kit were obtained from Pharmacia AB, Uppsala, Sweden. Blue-starch substrate was a gift from Pharmacia Diagnostica AB, Uppsala, Sweden. Schardinger-l~-dextrin ([Lcyclodextrin, cycloheptaamylose) and oyster glycogen were obtained from Sigma, St. Louis, USA. Anti-rabbit antibodies from swine and FITC-labelled anti-rabbit IgG antibodies from swine were obtained from Dakopatts A / S , Denmark. Monospecific antibarley-[~-amylase was a kind gift from Mr. E. D. Lund and Dr. J.. Hejgaard, The Danish Polytechnical University, Lundtofte, Denmark. Freund's complete and incomplete adjuvans were obtained from Behringwerke, Marburg, Germany. {~-limit dextrin was prepared from Linter starch (E. Merck, Darmstadt, Germany) by the method of UMEK1 and YAMAMOTO (22). [~-amylase (aamylase free) was obtained from Serva, Heidelberg, Germany. The resulting [~-limit dextrin was kindly analysed on the Sacchromat by Drs. B.S. ENEVOLDSENand F. SCHMIDTusing the method of SCHmDT and ENEVOLDSEN(19). Sacchromat analysis revealed that the product contained 83 % [Llimit dextrin components. Purified glutaraldehyde was the kind gift of Mr. E. LUND (Polytechnical University, Denmark) and of Drs. S. RASMUSSENand P. HYDE (Carlsberg Laboratory, Denmark). All other chemicals were Analytical Reagent grade. 2.2. Plant Material Seeds of Hordeum vulgare L. (cv. Nordal) were obtained from Dr. J. LADEN, Carlsberg Plant Breeding, Denmark. Barley green-malt was prepared on a rotating-drum laboratory unit. Seeds were steeped for 48 hours with two aeration periods of 8 hours, followed by 7 d of germination in the rotating drum unit at 15 ~ and 98 % r.h. Germinating seed material for the immunochemical localisation experiments was obtained by pressing seeds into moist sand and germinating at 15 ~ (98 % relative humidity) for the appropriate time. 2.3. Immunoelectrophoresis Rocket immunoelectrophoresis and crossed immunoelectrophoresis were performed according to WEEKE (23) using an electrophoresis buffer containing 0.073 M-Tris/HC1 0.024 Mdiethylbarbituric acid and 0.00035 M-Ca-lactate (final pH 8.6). Gels were directly stained in coomassie blue R 250 according to WEEKE(23), except in the cases where amylase measurement was carried out using the method of HEJGAARD (7). 2.4. Measurement of amylase isoenzymes Samples of column fractions were loaded into 20 !al rectangular wells on a 1.5 x 100 100 m m agarose gel containing electrophoresis buffer (see 2.3). The gel was subjected to a voltage of 12 V 9 cm -j for 1 hour. After drying in a cold air stream the gels were placed in a solution containing 1% lintner starch in 0.02 M-Na acetate buffer pH 5.5 for 1 hour at 37 ~ The gel was stained in a solution containing 0.008 M~ I2 and 0.5 M-KI for 30 sec. The amylase activity was identified as a white zone on the pinkishbrown background. 2.5. Purification of a-amylase Barley a-amylase was purified using the principles of SILVANOVlCHand HILL (20) with several modifications as follows: Barley greenmalt (cv. Nordal) was freeze-dried and milled on a Udy Cyclotec laboratory mill using a 1 mm sieve (Tecator AB, Sweden). The resulting flour was extracted by stirring in 1:3/w:v 0.2 M-Na-acetate buffer, pH 5.5 containing 0.001 M-CaC12 for 1 hour at 4 ~ Following centrifugation at 16.000 g for 15 min at 0 ~ the supernatant was filtered through 4 layers of Miracloth (Calbiochem, USA). Ethanol was added to 40 % v:v and the mixture was stirred for 1 hour at 4 ~ After centrifugation at 19.000 g for 30 min at 0 ~ the supernatant was treated with 2% w:v aqueous oyster glycogen to a final concentration of 1.88 mg - ml -~ supernatant. The ethanol concentration was readjusted to 40% v:v and following stirring for 1 hour at 4 ~ the glycogen precipitated crude a-amylase was collected by centrifugation at 48.000 g, for 15 min at 0 ~ The precipitate was dissolved in a small volume of extraction buffer and dialysed against the same buffer overnight at 4 ~ Undissolved material was removed by centrifugation (48.000 x g 15 min, 0 ~ and the supernatant applied to a column of cycloheptaamylose-substituted epoxy-activated sepharose 6B (prepared as described by SILVANOVICHand HILL (20)). Non-bound protein was removed from the column by washing in 0.2 M-Na acetate buffer, pH 5.5, containing 0.001 M-CaCI2. The aII isoenzyme was eluted in 10 mg 9ml -I [~-limit dextrin which was predissolved in boiling water and then added to concentrated buffer in order to obtain the desired final concentration. The aamylase remaining on the column was eluted with 8 mg 9 ml -I cycloheptaamylose in elution buffer. Following each run the column was washed in 1.5 M-NaC1 in water and reequilibrated in start buffer. Fractions were (...truncated)