Abstract
The present study performed the isolation of cytosolic ascorbate peroxidase (APX) isozymes and analyzed the pattern of their activity development and also investigated the change in some other enzyme activities related to the ascorbate-glutathione pathway from the senescing wheat leaves. The aim of this work is to examine the possibility that in the cytoplasm of wheat leaves the ascorbate-glutathione pathway p!ays a significant role in relation to leaf senescence involving an $H_2O_2$ accumulation and then to show the effect of benzyladenine (BA) on that pathway. During the leaf senescence characterized by increases in ChI breakdown and H202 accumulation under the 4-day dark incubation of matured leaf segments; i) no significant increase of total cytosolic APX was observed, ii) a dehydroascorbate reductase (DHAR) activity was decreased rapidly, iii) a slight increase of glutathione reductase (GR) activity occurred. In the BA-treated leaves; however, i) the total activity of APX increased conspicuously, ii) the decrease of DHAR activity was relatively inhibited, iii) the GR activity increase was more enhanced, and iv) the decrease of ascorbate content and the increase of H202 content were retarded as compared with those of control leaves. Three isozymes of cytosolic APX were found by using a native-electrophoretic gel in senescing wheat leaves and two of them occurred with major activity. In the developmental patterns of cytosolic APX isozymes, only two isozyme bands ("a" and "b") appeared with almost constant activity through 4 days of incubation in the control leaves, while one additional weak isozyme band ("c") and a little increase of "b" isozyme activity were detected in the BA-treated leaves. EspeciaUy, the development of "a" isozyme activity increased remarkably compared with that of control leaves. The increased capacity for peroxide scavenging due to the enhanced activity of all 3 enzymes (APX, DHAR, GR) participating in the ascorbate-glutathione pathway in BA-treated leaves suggested that this pathway might playa significant role in the processes related to the wheat leaf senescence.scence.
본 연구는 $H_2O_2$ 축적이 수반되는 잎의 노쇠현상과 관련하여 세포질내 ascorbate-glutathione 회로의 역할 가능성과 이때 benzyladenine(BA)의 효과를 밝히기 위하여 노쇠중인 밀 잎에서 시토졸 ascorbate peroxidase(APX) isozyme 분리 및 발달양상과 ascorbate-glutathione 회로 구성효소들의 활성도 변화를 조사하였다. 성숙한 밀 제1엽 절편을 4일간 암배양하는 동안 증가된 엽록소 분해 및 $H_2O_2$ 축적으로 규정되는 잎의 노쇠발달중 대조구 잎에서는 시토졸 APX 활성도의 유의성 있는 증가가 관찰되지 않았으며 dehydroascorbate reductase(DHAR)의 활성도는 급격히 감소되었고, glutathione reductase(GR) 활성도는 완만하게 증가하였다. 그러나 BA로 처리된 잎에서는 시토졸 APX 활성도가 현저하게 증가하였으며 DHAR 활성도의 감소가 지연되어 나타났고 GR 활성도의 증가는 대조구에 비해 증진되었으며 내재성 ascorbate 함량의 감소율과 H2O2 축적이 억제되었다. 3개의 시토졸 APX isozyme이 native-PAGE법에 의해 노쇠중인 밀 잎에서 발견되었으며 그 중 2개 isozyme은 높은 활성도를 보였다. 시토졸 APX isozyme의 발달양상의 경우 4일간의 암배양 동안 대조구 잎에서는 단지 2개의 isozyme band("a"와 "b")가 거의 같은 활성도를 지닌 채 출현하였으나 BA로 처리된 잎에서는 추가로 1개의 약한 isozyme band("c")가 더 나타났으며 "b" isozyme의 활성도가 약간 촉진되었다 그러나 "a" isozyme 활성도는 대조구 잎에 비해 암배양시간 경과에 따라 현저하게 발달하였다. 대조구 잎과 비교시 BA로 처리된 잎에서는 시토졸 APX isozyme의 발달 및 APX, DHAR, GR의 전체 활성도가 함께 증진되어 그 결과 $H_2O_2$ 제거능력이 증대된 본 실험의 결과는 ascorbate-glutathione 회로가 밀 잎의 노쇠과정에 중요하게 작용하고 있음을 제시하였다.
