Siminis C. I., Kanellis A. K., Roubelakis-Angelakis K. A.
Department of Biology, University of Crete, P.O. Box 1470, 71110 Heraklio, Crete, Greece (C.I.S., K.A.R.-A).
Plant Physiol. 1994 Aug;105(4):1375-1383. doi: 10.1104/pp.105.4.1375.
Based on our previous results that peroxidase is induced in dividing tobacco protoplasts but it is not expressed in the nondividing grapevine (Vitis vinifera L.) protoplasts during culture (C.I. Siminis, A.K. Kanellis, K.A. Roubelakis-Angelakis [1993] Physiol Plant 87: 263-270), we further tested the hypothesis that oxidative stress may be implicated in the recalcitrance of plant protoplasts. The expression of catalase, a major defense enzyme against cell oxidation, was studied during isolation and culture of mesophyll protoplasts from the recalcitrant grapevine and regenerating tobacco (Nicotiana tabacum L.). Incubation of tobacco leaf strips with cell wall-degrading enzymes resulted in a burst of catalase activity and an increase in its immunoreactive protein; in contrast, no such increases were found in grapevine. The cathodic and anodic catalase isoforms consisted exclusively of subunits [alpha] and [beta], respectively, in tobacco, and of subunits [beta] and [alpha], respectively, in grapevine. The catalase specific activity increased only in grapevine protoplasts during culture. The ratio of the enzymatic activities to the catalase immunoreactive protein declined in dividing tobacco protoplasts and remained fairly constant in nondividing tobacco and grapevine protoplasts during culture. Also, in dividing tobacco protoplasts the de novo accumulation of the catalase [beta] subunit gave rise to the acidic isoenzymes, whereas in nondividing tobacco and grapevine protoplasts, after 8 d in culture, only the basic isoenzymes remained due to de novo accumulation of the [alpha] subunit. The pattern of catalase expression in proliferating tobacco leaf cells during callogenesis was similar to that in dividing protoplasts. The different responses of catalase expression in dividing and nondividing tobacco and grapevine mesophyll protoplasts may indicate a specificity of catalase related to induction of totipotency.
基于我们之前的研究结果，即过氧化物酶在分裂的烟草原生质体中被诱导，但在培养过程中未分裂的葡萄（Vitis vinifera L.）原生质体中不表达（C.I. Siminis、A.K. Kanellis、K.A. Roubelakis-Angelakis [1993] Physiol Plant 87: 263 - 270），我们进一步检验了氧化应激可能与植物原生质体难再生有关的假说。在从难再生的葡萄和再生烟草（Nicotiana tabacum L.）中分离和培养叶肉原生质体的过程中，研究了过氧化氢酶（一种对抗细胞氧化的主要防御酶）的表达。用细胞壁降解酶处理烟草叶条导致过氧化氢酶活性激增及其免疫反应性蛋白增加；相比之下，在葡萄中未发现此类增加。在烟草中，阴极和阳极过氧化氢酶同工型分别仅由α和β亚基组成，而在葡萄中分别由β和α亚基组成。过氧化氢酶比活性仅在葡萄原生质体培养期间增加。在培养过程中，分裂的烟草原生质体中酶活性与过氧化氢酶免疫反应性蛋白的比率下降，而未分裂的烟草和葡萄原生质体中该比率保持相当恒定。此外，在分裂的烟草原生质体中，过氧化氢酶β亚基的从头积累产生了酸性同工酶，而在未分裂的烟草和葡萄原生质体中，培养8天后，由于α亚基的从头积累，仅保留了碱性同工酶。愈伤组织形成过程中增殖的烟草叶细胞中过氧化氢酶的表达模式与分裂的原生质体相似。分裂和未分裂的烟草及葡萄叶肉原生质体中过氧化氢酶表达的不同反应可能表明过氧化氢酶与全能性诱导相关的特异性。
