Evaluation of Roles of Amidase Which Converts Indole-3-Acetamide to Indole-3-Acetic Acid, in Formation of Clubroot in Turnip
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Amidase was investigated if it was one of auxin-producing enzymes of Brassica rapa. We found amidase activity to convert indole-3-acetamide to indole-3-acetic acid in soluble protein extracts from B. rapa. Optimum condition for the enzyme activity was searched and two optimum temperatures were obtained at 45 and 55℃. Search for the heat stability of the enzyme strongly suggested that the two summits of the optimum temperatures were resulted from occurrence of variant amidases showing different temperature stabilities. The possibility of the presence of several amidase isoforms was supported by the following two observations, i.e., firstly, amidase activities at 45 and 55℃ had different pH optimums, 8.5 and 7.5, respectively. Secondly, the enzyme activities at the two temperatures were differentially fluctuated during the vegetative growth of turnip and clubroot development. Fluctuation of the amidase activity and IAA contents observed in various turnip tissues such as healthy turnip leaf, hypocotyl and roots suggested that the enzyme had a constitutive role for keeping IAA homeostasis in those tissues. Interestingly, the amidase in turnip was shown to have high activity in hypocotyl and root rather than leaf, unlike Arabidopsis one. Changes of the enzyme activity during development of B. rapa was analyzed using the tissues of clubrootdiseased turnips. The activities fluctuated differently from each other in the temperature at 45 and 55℃ in infected tissues. In addition, activity at 45℃ was specifically enhanced in a later phase of clubroot development, whereas one at 55℃ increased only in an early phase in the infected root tissues. These results indicated that the amidase played an important role in turnip growth and clubroot development. 根こぶ病菌Plasmodiophora brassicae による根こぶ形成の分子機構解明を目的にオーキシン合成酵素の1つアミダーゼ活性を、カブ組織から可溶性粗酵素画分を調製し測定した。活性最適温度は45及び55℃であり、最適pH は、反応温度45℃でpH 8.5、55℃でpH 7.5付近であった。熱安定性を調べる為、粗酵素画分を45または55℃で加熱し、その後45及び55℃の反応温度で残存活性を測定した。45℃熱処理では、両測定温度で失活パターンは同一であった。55℃熱処理では、45℃での活性は70% 失活し、55℃では60％の活性が残存した。発芽後20～40日の間５日毎に健全カブと根こぶ病感染カブの葉、胚軸、根のアミダーゼ活性とIAA 含量を測定し根こぶ病とアミダーゼの関係を調べた。健全カブでは、上記全組織で安定なアミダーゼ活性があり恒常的な機能が推測された。一方感染カブの根で初期に55℃での活性が、後期には45℃の活性がそれぞれ健全カブより増加した。本研究で、複数のアミダーゼが存在し、それらが独立で根こぶ病発現に関与する可能性が初めて示唆された。
- 新潟大学農学部の論文
- 2007-08-00
新潟大学農学部 | 論文
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