Amylolytic Activity in Selected Sweetpotato ( Ipomoea batatas Lam) Varieties during Development and in Storage

Abstract

Sweetpotato varieties (five) were investigated for changes in α- and β-amylase activities during root development and on subjection of Agric. Biol. Chem., 49 (3), 737~744, 1985 737 Major Soluble Proteins of Sweet Potato Roots and Changes in Proteins after Cutting, Infection, or Storage He-sheng Li* and Kazuko Oba Laboratory oj Biochemistry, Faculty of Agriculture, Nagoya University, Chigusa-ku, Nagoya 464, Japan Received August 10, 1984 Study of the soluble proteins of sweet potato (Ipomoea batatas L. cv. Norin 1) roots showed that the major protein had an apparent molecular weight of25,000, and accounted for 60~ 70% of the total soluble protein extracted from fresh tissue. The 25-kDa protein exists in two forms, which can be resolved into two bands by nondenaturing polyacrylamide gel electrophoresis. Immunodif- fusion and crossed immunoelectrophoresis showed that these forms are immunologically identical. This protein was identified as the antigenic component A of sweet potato root.1} It was degraded to proteins of lower molecular weight (9,500 to 20,000) if the tissue was cut or infected by Ceratocystis fimbriata. As almost none of this 25-kDa protein was detected in roots stored for one year at 10~ 12°C, it is probably the storage protein of these roots. Another major protein was identified as /?-amylase by immunodiffusion and immunoelectrophoresis. The amount of jS-amylase did not change appreciably after cutting or infection, but it was present in only trace amounts in the roots stored for one year, Cutting, infection, or storage of root tissue resulted in the production of new isozymes ofperoxidase, acid phosphatase, and esterase. Increases in some other proteins in cut and in diseased tissues were detected by gel electrophoresis. The soluble proteins of sweet potato roots were studied in relation to metabolic alter- ations in response to pathogenic infection. Uritani & Stahmann1'2) reported that certain antigenic components are produced in sweet potato root tissue in response to infection by Ceratocystis fimbriata, and designated two of them as components B and D. Other major components, A and C, are present in all tissue extracts of fresh, cut, or diseased tissues of many varieties of sweet potato. Components C and D have been identified as /?-amylase and peroxidase A, respectively.1 3) However, the nature of component A, which is present in higher concentrations than most other anti- genic components of the extracts, and com- ponent B, which appears in diseased tissue in amounts roughly parallel to the magnitude of the resistant reaction of the host tissue, has not been elucidated. We examined the soluble pro- teins of sweet potato roots using polyacryl- amide gel electrophoresis and immunochemi- cal techniques. Little is known about the constituent pro- teins of most root and tuber storage organs, except for those of potatoes and yams, which have been studied in detail.4~9) Here, we found a major soluble protein that was degraded into peptides of lower molecular weight after stor- age, cutting, or fungal infection of the roots. MATERIALS AND METHODS Plant materials. Roots of sweet potato (Ipomoea ba- tatas, L. cv. Norin 1) harvested in autumn were stored at 10~ 12°C until use. The roots were surface-sterilized with 0. 1 % (v/v) sodium hypochlorite and the parenchymatous tissue was sliced 2 cm thick. Individual roots were cut into two parts. One part was inoculated with a spore suspen- sion (1 x 107/ml) of Ceratocystisfimbriata Ell. & Halst and incubated at 30°C for 44hr. Sections (l~2mm thick) * Present address: Department of Plant Physiology, Hua-zhong Agriculture Institute, Wuhan, The People 's Republic of China. 738 H.-s. Li and K. Oba from the non-infected tissue just below the infected part were used as diseased tissue. The other part was incubated under the same conditions without inoculation. Here sections (l~2mm) were taken from the tissue 0.5mm below the surface and used as cut tissue. Parenchymatous tissue of whole roots not subjected to inoculation and incubation was used as fresh tissue. Parenchymatous tissue of whole roots stored at 10~ 12°C for one year was used as stored tissue. Extraction of soluble proteins from tissues. The tissues (60g) were homogenized with 120ml of 0.05 m potassium phosphate buffer (pH 7.5) containing 0.5 m sucrose, 10mM MgCl2, 1% (w/v) ascorbic acid, and 24g ofPolyclar AT in a homoblender for 1 min. During the homogenization, the pH was adjusted to 7.5 with 10% (w/v) K2CO3. The homogenate was filtrered through two layers of nylon gauze and centrifuged at 9,000 x g for 20min. The super- natant was passed through a Sephadex G-25 column (4.5cm x 40cm) equilibrated with 0.05 m potassium phos- phate buffer (pH 7.5) containing 0.25m sucrose, 1mM MgCl2, and 0.5% (w/v) ascorbic acid, to remove poly- phenols from the solution. The effluent was centrifuged at 100,000 xg for 2hr, and the proteins in the supernatant were precipitated with ammonium sulfate (56. 1 g/100 ml). The precipitate was suspended in 0.05 m potassium phos- phate buffer (pH 7.5) containing 12.5% (v/v) glycerol and 0.1% (w/v) ascorbic acid, and dialyzed against the same buffer for 15hr with one buffer change, and then for 5 hr against buffer of the same composition but with 0.05% (w/v) ascorbic acid. The dialyzate was stored frozen at -20°C until use. Polyaerylamide gel electrophoresis. Sodium dodecyl sul- fate (SDS) polyacrylamide gel electrophoresis was done according to Laemmli10) on a 1-mm thick slab of 14.5% (w/v) polyacrylamide gel. Protein samples were denatured before electrophoresis by incubation for 20 min at 70°C in 1 % (w/v) SDS containing 1 % (v/v) 2-mercaptoethanol and 5% (w/v) sucrose. Nondenaturing polyacrylamide gel elec- trophoresis was done according to Davis11} on a 1-mm thick slab of 7.5% (w/v) polyacrylamide gel. The current was a constant 10mA/cm2. The separated proteins were stained with Coomassie Blue R 250 (0.25% in MeOH-HOAc-H2O, 5 : 1 : 4, v/v/v). Densitometric anal- ysis of the stained gels was done on a Toyo digital Densitorol (Model DMU-33C). Zymograms of peroxidase, acid phosphatase, and es- terase were prepared by staining the gels according to the following procedure. Peroxidase activity was located by staining the gel for 15min at 25°C in 0.1 m citrate buffer (pH 5.0) containing o-phenylenediamine (0.4 mg/ml) and 0.01% (v/v) H2O2.12) Acid phosphatase activity was lo- cated by staining the gel for 1 hr at 25°C in 0.05m acetate buffer (pH 5.0) containing 1 mM 1-naphthyl phosphate and Fast Garnet GBC salt (0.5 mg/ml).13) Esterase activity was located by staining the gel for 20min at 25°C in 0.1m phosphate buffer (pH 7.0) containing 5mM 1-naphthyl acetate, 5 niM 2-naphthyl acetate, and Fast Blue RR salt (1 mg/ml).14) Quantitative evaluation of major proteins. The ratio of the amounts of major proteins to the total soluble proteins was determined from quantitative measurements of the amount of dye bound to each protein, obtained by integrating the densitometric scans of gels after SDS polyacrylamide gel electrophoresis. Preparation ofantisera to soluble proteins. Samples each of 15 mg of the total soluble proteins extracted from fresh and diseased tissues were mixed with an equal volume of complete Freund's adjuvant. The mixture was injected twice subcutaneously into a rabbit at an interval of 15 days. Seven days after the second injection, lOmg of each protein was mixed with an equal volume of incomplete adjuvant and this mixture was injected subcutaneously. The rabbit was bled 9 days after the final injection. Immunochemical tests. Double immunodiffusion tests were carried out as described by Ouchterlony and Nilson15) on a 1% (w/v) agarose plate containing 0.01 m phosphate buffer (pH 7.2), 0.15m NaCl, and 0.1% (w/v) NaN3. The dishes were kept in a moist chamber at 25°C. Crossed immunoelectrophoresis was also done accord- ing to Ouchterlony and Nilson. First, soluble proteins from fresh and diseased tissues were individually separated by nondenaturing polyacrylamide gel electrophoresis on a 1-mm thick slab of7.5% (w/v) gel (pH 8.9). Then a strip of this gel (6mm wide) was mounted on a 1% (w/v) agarose plate (5 x 5cm) containing both barbital-HCl buffer (pH 8.6, /i=0.02) and 3% (v/v) antiserum to total soluble proteins from diseased tissue or to band 9 protein (25- kDa) purified by Dr. M. Maeshima of this laboratory (data will be published elsewhere). A constant current of 5 mA was applied to the plate for 15 hr. After immunoelec- trophoresis, the plate was washed and stained with Coomassie Blue R 250. Protein Determination. Proteins in tissue extracts were precipitated with 10% (w/v) trichloroacetic acid (TCA), dissolved in 1 n NaOH, and analyzed according to Lowry et al.16) with bovine serum albumin as the reference protein. RESULTS AND DISCUSSION Analysis of proteins by polyacrylamide gel elec trophoresis Uritani & Stahmann1* separated the soluble proteins of sweet potato roots into several bands by starch gel electrophoresis, but with- out good resolution. We tried to separate them by SDS and nondenaturing polyacryl- Major Soluble Proteins of Sweet Potato Roots 739 n F C 0 Fo 94k Illll iili iiiii iiiiiiiii iiSil llill Fig. 1. SDS Polyacrylamide Gel Electrophoresis of Total Soluble Proteins from Fresh (F), Cut (C), Diseased (D), and Stored (Fo) Sweet Potato Root Tissues. Each lane was loaded with 100/ig of protein. Molecular weight markers appear in lane M. amide gel electrophoresis. SDS polyacryl- amide gel electrophoresis separated the pro- teins offresh roots into one major band, a few prominent bands, and many minor bands (lane F in Fig. 1). The major protein had an ap- parent molecular weight of 25,000 and was designated the 25-kDa protein. Its amount was less while that of lower-molecular-weight pro- teins in the range of 9,500 to 20,000 was greater in cut and diseased tissue extracts (lanes C and D in Fig. 1). A very different pattern of proteins was found in tiss