KOREAN JOURNAL OF CROP SCIENCE (한국작물학회지)

The Korean Society of Crop Science (한국작물학회)
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Biochemical and Ultrastructural Trends in Proteolysis of the $\beta$-subunit of 7S Protein in the Cotyledons During Germination of Soybean Seeds

  • Krishnan, Hari B. (USDA-ARS, Plant Genetics Research Unit and Department of Agronomy, University of Missouri)
  • Published : 2002.06.01
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Abstract

Antibodies raised against the purified p-subunit of $\beta$-conglycinin were used in immunohistochemical studies to monitor the pattern of $\beta$-conglycinin mobilization in the cotyledons during soybean [Glycine max (L.) Merr.] seed germination. Western blot analysis revealed that the break down of the $\beta$-subunit of $\beta$-conglycinin commenced as early as 2 days after seed imbibition (DAI). Concurrent with the degradation of the $\beta$-subunit of $\beta$-conglycinin, accumulation of 48, 28, and 26 kD proteolytic intermediates was observed from 2 to 6 DAI. Western blot analysis also revealed that the acidic subunit of glycinin was mobilized earlier than the basic subunit. The basic glycinin subunit was subjected to proteolysis within 2 DAI resulting in the appearance of an intermediate product approximately 2 kD smaller than the native basic glycinin subunit. In contrast to the major seed storage proteins, lipoxygenase was subjected to limited proteolysis and was detected even after 8 DAI. The first sign of $\beta$-conglycinin breakdown was observed near the vascular strands and proceeded from the vascular strands towards the epidermis. Protein A-gold localization studies using thin sections of soybean cotyledons and antibodies raised against the $\beta$-subunit of $\beta$-conglycinin revealed intense labeling over protein bodies. A pronounced decrease in the protein A-gold labeling intensity over protein bodies was observed at later stages of seed germination. The protein bodies, which were converted into a large central vacuole by 8 DAI, contained very little 7S protein as evidenced by sparse protein A-gold labeling in the vacuoles.

Keywords

References

  1. Briarty, L. G., D. A. Coult, and D. Boutler. 1970. Protein bodies of germinating seeds of Vwia faba. Changes in fine structure and biochemistry. J. Exp. Bot. 21 : 513-524 https://doi.org/10.1093/jxb/21.2.513
  2. Catsimpoolas, N., T. G. Campbell, and E. W. Meyer. 1968. Immu-nochemical study of changes in reserve proteins of germinating soybean seeds. Plant Physiol. 43 : 799-805 https://doi.org/10.1104/pp.43.5.799
  3. Harris, N., and M. A. Chhspeels. 1975. Histochemical and biO-chemical observation on storage protein metabolism and Pro-tein body autolysis in cotyledons of germinating mung beans. Ptant Physiol. 56 : 292-299 https://doi.org/10.1104/pp.56.2.292
  4. Hartl, P. M., A. L. TanWilson, and K. A. Wilson. 1986. Proteolysis of Kunitz soybean trypsin inhibitor during germination. Phytochemistry 25:23-26 https://doi.org/10.1016/S0031-9422(00)94494-7
  5. Hayat, M. A. 1972. Basic electron microscopy techniques, Van Nostrand Reinhold, New York
  6. Hohsberger, M. M.-F. Clerc, and J.-J. Pahud, Ultrastructural local-ization of glycinin and B-conglycinin in Gtycine max (soybean)cv. Maple Arrow by the immunogold method. Histochemistry 85(1986) : 291-294 https://doi.org/10.1007/BF00493479
  7. Khshnan, H. B. 1999. Characterization of high-lysine mutants of rice. Crop Sci. 39: 825-831 https://doi.org/10.2135/cropsci1999.0011183X003900030035x
  8. Khstman, H. B. and T. W. Okita. 1896. Structural relationship among the rice glutelin polypeptides, Plant Physiol. 81 : 748-753 https://doi.org/10.1104/pp.81.3.748
  9. hshnan, H. B., V. R. Franceschi, and T. W. Okita. 1986. ImmU-nochemical studies on the role of the Golgi complex in protein body formation in rice seeds. Ptanta 169 : 471-480
  10. Krishnan, H. B., G. Jiang, A. H. Krishnan, and W. J. Wiebold. 2000. Seed storage protein composition of non-nodulating Soy-bean (Glycine max [L.] Merr.) and its influence on protein qual-ity. Plant Sci. 157 : 191-199 https://doi.org/10.1016/S0168-9452(00)00283-1
  11. Laemmli, U. K. 1970. Cleavage of structural proteins during assembly ofphage T4 head. Nature Ill : 680-685
  12. Madden, M. A., A. L. Tan-Wilson, and K. A. Wilson. 1985. Pro-teolysis of soybean Bowman-Birk trypsin inhibitor during ger-initiation. Phytochemistry 24 : 2811-2815 https://doi.org/10.1016/0031-9422(85)80005-4
  13. Melroy, D. L., and E. M. Herman. 1991. TIP, an integral mem-brane protein of the protein-storage vacuoles of the soybean cotyledon undergoes developmentally regulated membrane accumulation and removal. Planta 184 : 113-122
  14. Muntz, K. 1996. Proteases and proteolytic cleavage of storage Pro-terns in developing and germinating dicotyledonous seeds. J.Exp. Bot.47 : 605-622 https://doi.org/10.1093/jxb/47.5.605
  15. Nielsen, N. C. 1996. Soybean seed composition, In: D.P.S. Venna, R. C. Shoemaker (Eds.) Soybean Genetics, Molecular Biology and Biotechnology. CAB International, Wallingford, Oxon, UK,PP. 127-163
  16. Okita, T. W., H. B. Krishnan, and W. T. Kim. 1988. Immunologi-cal relationships among the major seed proteins of cereals, Plant Sci. 57 : 103-111 https://doi.org/10.1016/0168-9452(88)90075-1
  17. $\ddot{O}pik$, H. 1966. Changes in cell fine structure in the cotyledons of Phaseolus vulgaris L. during germination. J. Exp. Bot. 17 : 427-439 https://doi.org/10.1093/jxb/17.3.427
  18. Qi, X., K. A. Wilson, and A. L. Tan-Wilson. 1992. Characteriza-tion of the major protease involved in the soybean $\beta-$conglyci-nin storage protein mobilization. Plant Physiol. 99 : 725-733 https://doi.org/10.1104/pp.99.2.725
  19. Schlereth, A- D. Standhardt, H-P. Mock, and K. Muntz. 2001. Stored cysteine proteinase start globulin moblization in protein bodies of embryonic axes and cotyledons during vetch (Vicia sativa L.) seed germination. Planta 212 : 718-727 https://doi.org/10.1007/s004250000436
  20. Sebastiani, F. L., L. B. Farrell, M. A. Schuler, and R. N. Beachy. 1990. Complete sequence of a cDNA of the $\alpha-$subunit of Soy-bean $\beta-$conglycininin. Ptant Mol. Biol. 15 : 197-201 https://doi.org/10.1007/BF00017745
  21. Seo, S., A. Tan-Wilson, and K. A. Wilson. 2001. Protease C2, a cysteine endopeptidase involved in the continuing mobilization of soybean $\beta-$conglycmin seed proteins. Biochim. Biophys .Acta 154: 192-206
  22. Sheehan, D. C, and B. B. Hrapchak. 1980. Theory and practice of histotechnology, Battelle Press, Columbus, Ohio
  23. Smith, O. L., and A. M. FIinn. 1967. Histology and histochemistry of the cotyledons of Pisum arvense L. during germination. Planta 74: 72-85 https://doi.org/10.1007/BF00385172
  24. Spurr, A. P. 1969. A low-viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res. 26 : 31-43 https://doi.org/10.1016/S0022-5320(69)90033-1
  25. Tiedemann, J., B. Neubohn, and K. Muntz. 2000. Different func-tions of vicilin and legumin are reflected in the histopattem of globulin mobilization during germination of vetch (Vwia sativa L.). Planta 211: 1-12 https://doi.org/10.1007/s004250000259
  26. Tierney, M. L., E. A. Bray, R. D. Allen, Y. Ma, R. F. Drong, J. Slishtom, and R. N. Beachy. 1987. Isolation and Characteriza-tion of a genomic clone encoding $\beta-$subunit $\beta-$conglyci-nin .Planta 172: 356-363 https://doi.org/10.1007/BF00398664
  27. Wilson, K. A. 1986. Role of proteolytic enzymes in the mobiliza-tion of protein reserves in the germinating dicot seed. In: M. J. Dalling (Ed.), Plant Proteolytic Enzymes, vol n. CRC Press Inc., Boca Raton, Florida, pp. 19-47
  28. Wilson, K. A., B. R. Rightmire, J. C. Chen, and A. L. Tan Wilson. 1986. Differential proteolysis of glycinin and B-conglycinin polypeptides during soybean germination and seedling growth. Plant Physiol. 82: 71-76 https://doi.org/10.1104/pp.82.1.71
  29. Wilson, K. A., G. Papastoitsis, P. Hartl, and A. L. Tan-Wilson. 1988. Survey of the proteolytic activities degrading the Kunitz trypsin inhibitor and glycinin in germinating soybeans (Glycine max). Plant Physiol. 88 : 355-360 https://doi.org/10.1104/pp.88.2.355