Study of relationship between development of embryo specific endopeptidase activites and induction of seed germination in maize plant.

1998 Fiscal Year Final Research Report Summary

• Project/Area Number: 09660071
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: 応用微生物学・応用生物化学
• Research Institution: YAMAGATA UNIVERSITY
• Principal Investigator: MITSUHASHI Wataru YAMAGATA UNIVERSITY,DEPARTMENT OF BIORESOUCE ENGINEERING,ASSISTANT PROFESSOR, 農学部, 助教授 (50192761)
• Co-Investigator(Kenkyū-buntansha): TOYOMASU Tomonobu YAMAGATA UNIVERSITY,DEPARTMENT OF BIORESOUCE ENGINEERING,ASSISTANT PROFESSOR, 農学部, 助教授 (60272085)
• Project Period (FY): 1997 – 1998
• Keywords: ENDOPEPTIDASE / DRY SEED / EMBRYO / GERMINATION

Research Abstract

Very high proteinase activity was observed in maize (Zea mays L dentcorn cv. W64A) dry seeds. Major activity was shown as one band which has 0.45 of Rf value on a native PAGE after activity staining. The activity located specifically in embryo from dry seed, but not in endosperm and scutelum. The enzyme may have an important role during early stage of germination, because of the activity disappeared until 48 hours though the seed will germinate after 72 hours after imbibition. The role of this enzyme is interested in induction of seed germination, because timing of development of the activity is concomitant with degradation of LEA proteins in the embryo.
It was very hard to detect the activity stably in crude extract. Many effectors affected the activity in crude extract. For example, monovalent cation, especially, sodium ion, disappeared the activity dramatically. And, the activity increased significantly by treatment of EDTA.However, the activation may not due to the chelate metallo-ion. The effect of specific inhibitors for protease showed the enzyme is a trypsin-like serine-endopeptidase.
Sedimentation by Ammonium sulfate from crude extract was effectively to have it's stabale activity. Purification of the enzyme was attempted by using ion-exchange chromatography, gel-filtration, hydrophobic chromatography, and native PAGE.Combination of these chromatography always gave the same 15-20 peptides which have 45-65 kD of molecular masses on SDS-PAGE.Affinity chromatography also gave the same pattern of peptides. These results indicate that the enzyme may make a complex with these peptides at least in vitro.