Pastore D, Trono D, Laus M N, Di Fonzo N, Passarella S
Dipartimento di Scienze Animali, Vegetali e dell'Ambiente, Facoltà di Agraria, Università del Molise, Via De Sanctis, 86100 Campobasso, Italy.
Plant Cell Physiol. 2001 Dec;42(12):1373-82. doi: 10.1093/pcp/pce174.
In order to gain a first insight into the alternative oxidase (AO) function in durum wheat mitochondria (DWM), we investigated some activation pathways of this enzyme in DWM purified from both etiolated shoots and green leaves. AO was activated when DWM were added with either pyruvate, known as an AO activator in other plant mitochondria, or alanine plus 2-oxoglutarate, which can generate intramitochondrial pyruvate and glutamate via transamination. In contrast, no AO activity was observed during oxidation of malate plus glutamate or succinate (which can generate malate). In this regard DWM differ from other plant mitochondria. Moreover, DWM were found: (i) to have a very low malic enzyme (ME) activity, (ii) to release oxaloacetate rather than pyruvate during malate oxidation and (iii) to poorly oxidise malate in the absence of glutamate, which removes oxaloacetate via transamination. Therefore, we show that, unlike other plant mitochondria, no pyruvate is generated inside DWM from malate via ME, allowing no AO activity. Other AO activators, alternative to pyruvate, were checked by evaluating the capability of several compounds to induce oxygen uptake and/or electrical membrane potential (Delta Psi) in cyanide-treated DWM. Hydroxypyruvate and glyoxylate, photorespiratory cycle intermediates, were found to be powerful AO activators, capable of inducing a maximal rate of cyanide-insensitive oxygen uptake 1.7 times and 2.3 times higher than pyruvate, respectively. These results suggest that in durum wheat a link may exist between AO activity and photorespiratory metabolism rather than malate metabolism. Moreover, we observed that AO activation resulted in both a partially coupled respiration and a reduction by half of the rate of superoxide anion generation; therefore, AO is expected to work as an antioxidative defence system when the photorespiratory cycle is highly active, as under environmental stress.
为了初步了解硬粒小麦线粒体（DWM）中交替氧化酶（AO）的功能，我们研究了从黄化苗和绿叶中纯化得到的DWM中该酶的一些激活途径。当向DWM中添加丙酮酸（在其他植物线粒体中被称为AO激活剂）或丙氨酸加2 - 氧代戊二酸时，AO被激活，丙氨酸加2 - 氧代戊二酸可通过转氨作用生成线粒体内的丙酮酸和谷氨酸。相反，在苹果酸加谷氨酸或琥珀酸（可生成苹果酸）的氧化过程中未观察到AO活性。在这方面，DWM与其他植物线粒体不同。此外，还发现DWM：（i）苹果酸酶（ME）活性非常低，（ii）在苹果酸氧化过程中释放草酰乙酸而非丙酮酸，（iii）在没有谷氨酸（通过转氨作用去除草酰乙酸）的情况下，苹果酸氧化能力较差。因此，我们表明，与其他植物线粒体不同，DWM中苹果酸不能通过ME生成丙酮酸，从而不会产生AO活性。通过评估几种化合物在氰化物处理的DWM中诱导氧气摄取和/或膜电位（ΔΨ）的能力，检查了除丙酮酸之外的其他AO激活剂。发现光呼吸循环中间体羟基丙酮酸和乙醛酸是强大的AO激活剂，能够分别诱导比丙酮酸高1.7倍和2.3倍的最大氰化物不敏感氧气摄取速率。这些结果表明，在硬粒小麦中，AO活性与光呼吸代谢而非苹果酸代谢之间可能存在联系。此外，我们观察到AO激活导致部分偶联呼吸以及超氧阴离子生成速率降低一半；因此，预计在环境胁迫等光呼吸循环高度活跃时，AO可作为一种抗氧化防御系统发挥作用。
