Abstract
α-Galactosidases (EC 3.2.1.22) from resting and germinated date (Phoenix dactylifera L.) seeds were compared and localized using immunocytochemical methods. The enzyme was present in both the endosperm and embryo of resting seeds, in the endosperm undergoing digestion where the greatest specific activity was present, and in the haustorium of seedlings. The enzyme had a molecular mass of 140000 as determined by gel filtration and a pH optimum of 4.5. At least seven forms of the enzyme with isoelectric points ranging from 3.85 to 5.2 were detected in the haustorium whereas only four of these forms were present in the endosperm. The relative activity levels of the various forms also differed between the two tissues. On Western blots all enzyme forms were recognized by antibodies raised against mung-bean (Vigna radiata) α-galactosidase. Using immunogold techniques, label was shown to be present in the protein bodies of the resting embryo cells but to decrease in this organelle as the reserve protein was mobilized and to appear diffusely in the cytoplasm in subsequent stages. In resting endosperm cells, label occurred in the protein bodies and in a thin region of inner wall. In endosperm undergoing digestion, where different stages of protoplast and wall breakdown occurred, immunogold staining was localized in the flocculent contents of vacuoles which resulted from storageprotein breakdown, then dense staining occurred in the inner wall of cell cavities formed by the complete dissolution of the cytoplasm, and finally, staining was uniformly diffuse throughout the remaining endosperm wall adjacent to the haustorium surface. These observations indicate that the α-galactosidase present in cell walls of the date palm endosperm during mannan mobilization is not secreted by the haustorium but instead is probably a pregermination product stored mainly in the protein bodies of resting endosperm and is released to the wall following loss of membrane integrity.
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Abbreviations
- IEF:
-
isoelectric focusing
- IgG:
-
immunoglobulin G
- Mr :
-
relative molecular mass
- pI:
-
isoelectric point
References
Alang, Z.C., Moir, G.F.J., Jones, L.H. (1988) Composition, degradation and utilization of endosperm during germination in the oil palm (Elaeis guineensis Jacq.). Ann. Bot. 61, 261–268
Balasubramaniam, K., Dey, P.M., Pridham, J.B. (1976) α-Galactosidase from coconut kernel. Phytochemistry 15, 1445–1446
Benjavongkulchai, E., Spencer, M.S. (1989) Barley aleurone xylanase: its biosynthesis and possible role. Can. J. Bot. 67, 297–302
Chandra Sekhar, K.N., DeMason, D.A. (1988) Quantitative ultrastructure and protein composition of date palm (Phoenix dactylifera) seeds: a comparative study of endosperm vs. embryo. Am. J. Bot. 75, 323–329
Chandra Sekhar, K.N., DeMason, D.A. (1989) Differential activity of acid phosphatases from the endosperm and haustorium of date palm (Phoenix dactylifera) seeds. Can. J. Bot. 67, 1096–1102
del Campillo, E., Shannon, L.M., Hankins, C.N. (1981) Molecular properties of the enzymic phytohemagglutinin of mung bean. J. Biol. Chem. 256, 7177–7180
DeMason, D.A. (1984) Growth parameters in the cotyledon of date seedlings. Bot. Gaz. 145, 176–183
DeMason, D.A. (1985) Histochemical and ultrastructural changes in the haustorium of date (Phoenix dactylifera). Protoplasma 126, 168–177
DeMason, D.A., Thomson, W.W. (1981) Structure and ultrastructure of the cotyledon of date palm (Phoenix dactylifera). Bot. Gaz. 142, 320–328
DeMason, D.A., Sexton, R., Reid, J.S.G. (1983) Structure, composition and physiological state of the endosperm of Phoenix dactylifera. Ann. Bot. 52, 71–80
DeMason, D.A., Sexton, R., Gorman, M., Reid, J.S.G. (1985) Structure and biochemistry of endosperm breakdown in date palm (Phoenix dactyliferd) seeds. Protoplasma 126, 159–167
DeMason, D.A., Chandra Sekhar, K.M., Harris, M. (1989) Endosperm development in the date palm (Phoenix dactyliferd) (Arecaceae). Am. J. Bot. 76, 1255–1265
DeMason, D.A., Stillman, J.I., Ellmore, G.S. (1989) Acid phosphatase localization in seedling tissues of the palms, Phoenix dactylifera and Washingtonia filifera, and its relevance to controls of germination. Can. J. Bot. 67, 1103–1110
Dey, P.M., del Campillo, E. (1984) Biochemistry of the multiple forms of glycosidases in plants. Adv. Enzymol. 56, 141–249
Dey, P.M., Pridham, J.B. (1972) Biochemistry of α-galactosidases. Adv. Enzymol. 36, 91–131
Dey, P.M., Wallenfels, K. (1974) Isolation and characterization of galactosidase from Lens esculenta. Eur. J. Biochem. 50, 107–112
Faye, L., Greenwood, G.S., Herman, E.M., Sturm, A., Chrispeels, M.J. (1988) Transport and posttranslational processing of the enzyme α-mannosidase in jack-bean cotyledons. Planta 174, 271–282
Fincher, G.B. (1989) Molecular and cellular biology associated with endosperm mobilization in germinating cereal grains. Annu. Rev. Plant Physiol. 40, 305–346
Foster, A.S., Gifford, E.M., Jr. (1974) Comparative morphology of vascular plants, 2nd edn. W.H. Freeman, San Francisco
Gubler, F., Ashford, A.E., Jacobsen, J.V. (1987) The release of α-amylase through gibberellin-treated barley aleurone cell walls: An immunocytochemical study with Lowicryl K4M. Planta 172, 155–161
Hankins, C.N., Shannon, L.M. (1978) The physical and enzymatic properties of a hemagglutinin from mung beans. J. Biol. Chem. 253, 7791–7797
Hankins, C.N., Kindinger, J.I., Shannon, L.M. (1980) Legume α- galactosidase forms devoid of hemagglutinin activity. Plant Physiol. 66, 375–378
Harlow, E., Lane, D. (1988) Antibodies: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, L.I., N.Y.
Harpaz, N., Flowers, H.M., Sharon, N. (1977) α-Galactosidase from soybeans destroying blood group B antigens. Eur. J. Biochem. 77, 419–426
Herman, E.M., Shannon, L.M. (1985) Accumulation and subcellular localization of α-galactosidase hemagglutinin in developing soybean cotyledons. Plant Physiol. 77, 886–890
Hughes, S.G., Overbeeke, N., Robinson, S., Pollock, K., Smeets, F.L.M. (1988) Messenger RNA from isolated aleurone cells directs the synthesis of an alpha-galactosidase formed in the endosperm during germination of guar (Cyamopsis tetragonaloba) seed. Plant Mol. Biol. 11, 783–789
Jones, R.L., Robinson, D.G. (1989) Protein secretion in plants. New Phytol. 111, 567–597
Keusch, L. (1968) Die Mobilisierung des Reservemannans im keimenden Dattelsamen. Planta 78, 321–350
McCleary, B.V., Matheson, N.K. (1974) α-d-Galactosidase activity and galactomannan and galactosylsucrose oligodisaccharide depletion in germinating legume seeds. Phytochemistry 13, 1747–1757
Meier, H. (1958) On the structure of cell walls and cell wall mannans from ivory nuts and from dates. Biochim. Biophys. Acta 28, 229–240
Mujer, C.V., Ramirez, D.A., Mendoza, E.M.T. (1984) Coconut α-galactosidase isoenzymes: Isolation, purification and characterization. Phytochemistry 23, 1251–1254
Opute, F.I. (1975) Lipid composition and the role of the haustorium in the young seedling of the West African oil palm, Elaeis guineensis. Ann. Bot. 39, 1057–1061
Peterson, G.L. (1977) A simplification of the protein assay method of Lowry et al., which is more generally applicable. Anal. Biochem. 83, 346–356
Reid, J.S.G., Meier, H. (1972) The function of the aleurone layer during galactomannan mobilization in germinating seeds of fenugreek (Trigonella foenum-graecum), crimson clover (Trifolium incarnatum L.) and lucerne (Medicago sativa L.): a correlative biochemical and ultrastructural study. Planta 106, 49–60
Reid, J.S.G., Meier, H. (1973) Enzymic activities and galactomannan mobilization in germinating seeds of fenugreek (Trigonella foenum-graecum, Leguminosae). Secretion of alpha galactosidase and beta mannosidase by the aleurone layer. Planta 112, 301–308
Righetti, P.G. (1983) Isoelectric focusing: theory, methodology, and applications. Elsevier, New York
Roth, J. (1983) The colloidal marker system for light and electron microscopic cytochemistry. In: Techniques in immunocytochemistry, vol. II. pp. 217–284, Bullock, G.R., Petrusz, P., eds. Academic Press, London
Seiler, A. (1977) Galaktomannanabbau im keimenden Johannisbrotsamen (Ceratonia siliqua L.). Planta 134, 209–221
Tomlinson, P.B. (1960) Essays on the morphology of palms. Principes 4, 56–61
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This study was funded by National Science Foundation grant PCM 8709813 to D.A.D. Mr. Jim I. Stillman did the electron microscopy. We thank Dr. C.N. Hankins for providing the antiserum used and for critical comments on the manuscript. We also thank Dr. W.W. Thomson for use of his electron microscope and for reading this manuscript. We extend our appreciation to Oasis Date Gardens, Coachella Valley, Cal., for a generous supply of date seeds.
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Sekhar, K.N.C., DeMason, D.A. Identification and immunocytochemical localization of α-galactosidase in resting and germinated date palm (Phoenix dactylifera L.) seeds. Planta 181, 53–61 (1990). https://doi.org/10.1007/BF00202324
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DOI: https://doi.org/10.1007/BF00202324