Characterization of Transformed Arabidopsis with Altered Alternative Oxidase Levels and Analysis of Effects on Reactive Oxygen Species in Tissue  

Abstract

The alternative oxidase (AOX) of plant mitochondria transfers electrons from the ubiquinone pool to oxygen without energy conservation. AOX can use reductant in excess of cytochrome pathway capacity, preventing reactive oxygen species (ROS) formation from an over-reduced ubiquinone pool, and thus may be involved in acclimation to oxidative stresses. The AOX connection with mitochondrial ROS has been investigated only in isolated mitochondria and suspension culture cells. To study ROS and AOX in whole plants, transformed lines of Arabidopsis (Arabidopsis thaliana) were generated: AtAOX1a overexpressors, AtAOX1a anti-sense plants, and overexpressors of a mutated, constitutively active AtAOX1a. In the presence of KCN, leaf tissue of either mutant or wild-type AOX overexpressors showed no increase in oxidative damage, whereas antisense lines had levels of damage greater than those observed for untransformed leaves. Similarly, ROS production increased markedly in anti-sense and untransformed, but not overexpressor, roots with KCN treatment. Thus, AOX functions in leaves and roots, as in suspension cells, to ameliorate ROS production when the cytochrome pathway is chemically inhibited. However, in contrast with suspension culture cells, no changes in leaf transcript levels of selected electron transport components or oxidative stress-related enzymes were detected under nonlimiting growth conditions, regardless of transformation type. Further, a microarray study using an anti-sense line showed AOX influences outside mitochondria, particularly in chloroplasts and on several carbon metabolism pathways. These results illustrate the value of expanding AOX transformant studies to whole tissues.The cyanide-resistant alternative oxidase (AOX) of plant mitochondria accepts electrons from the ubiquinone (UQ) pool and uses them to reduce oxygen to water, with no conservation of energy through proton gradient formation. AOX can compete with the energyconserving cytochrome (Cyt) pathway for reductant (Millenaar and Lambers, 2003;Finnegan et al., 2004), and in vivo studies using oxygen isotope fractionation indicate that AOX is nearly always active (Robinson et al., 1995). This situation raises the question of why an apparently energy-wasteful pathway operates in plant mitochondria.Because AOX can use reductant in excess of either the Cyt pathway capacity or the rate of ATP use, AOX may act to reduce formation of reactive oxygen species (ROS; Purvis and Shewfeldt, 1993;Millar et al., 2001a;Mittler, 2002). Over-reduction of the mitochondrial UQ pool is an important source of cellular ROS (Møller, 2001), and AOX, unrestricted by proton gradient size, can prevent such over-reduction. AOX function in this respect has been demonstrated in culture cells and isolated mitochondria. Tobacco (Nicotiana tabacum) culture cells anti-sense for AOX produce more ROS than wild-type cells, while AOX-overexpressing cells produce less (Maxwell et al., 1999). In isolated mitochondria, ROS production decreases when AOX is activated by a-keto acids (Past...

Mitochondrial complex II has a key role in mitochondrial-derived reactive oxygen species influence on plant stress gene regulation and defense

“…However, it has also been reported that succinate-dependent mH 2 O 2 production is faster than pyruvate/malate-dependent mH 2 O 2 production in plants, indicating that Complex II has a larger role than Complex I (40). Additionally, the ubiquinone pool itself has been highlighted as a site of ROS production in plant mitochondria (41).…”

Reactive Oxygen Species-Driven Transcription in Arabidopsis under Oxygen Deprivation

“…A, Regulation of ROS-related TF in the O 2 deprivation microarray experiments. B, Regulation of HsfA2, ZAT12, ZAT6, and ADH1 gene in wild-type and rbohD 2 plants under anoxia in a time course up to 8 h. A 6h 1 , 6 h of anoxia treatment without or with 90 mM Suc (s) added before the treatment (Loreti et al, 2005); A 6h 2 , 6 h of anoxia treatment (Banti et al, 2010); A 12h, 12 h of anoxia treatment, total and polysomal (p) RNA (Branco-Price et al, 2005); H 4h, 4 h of hypoxia treatment at 1% O 2 (Licausi et al, 2010); S 7/24h, 7 and 24 h of submergence treatment (Loreti et al, 2005); A 6h 2 , 6 h of anoxia treatment (Banti et al, 2010); A 12h, 12 h of anoxia treatment, total and polysomal (p) RNA (Branco-Price et al, 2005); H 4h, 4 h of hypoxia treatment at 1% O 2 (Licausi et al, 2010); S 7/24h, 7 and 24 h of submergence treatment ; flu 0.5 (30')/1/2h, flu mutant shift from dark to light (op den Camp et al, 2003); AOX1a-AS, AOX1a-AS mutant (Umbach et al, 2005) heat shock in Arabidopsis cell (Rikhvanov et al, 2007). Moreover, in mammals' pulmonary arteries hypoxiadriven mitochondrial ROS production has been suggested to trigger NADPH oxidase activity, suggesting a mechanism by which mitochondria and cytosol both contribute to the increase in ROS production during low O 2 (Rathore et al, 2008).…”

Photosynthesis and cell respiration modulated by water deficit in grapevine (Vitis vinifera L.) cv. Cabernet Sauvignon

“…This alternative pathway is nonphosphorylating, resistant to cyanide and antimicine and inhibited by salicilhydroxamic acid (SHAM) and n-propyl gallate (Schonbaum et al, 1971;Siedow and Grivin, 1980). It has been the focus of many studies in plant respiratory metabolism under several environmental stresses, mainly because an increased AOX capacity might contribute to controlling the formation of reactive oxygen species (ROS) (Wagner, 1995;Popov et al, 1997;Maxwell et al, 1999;Umbach et al, 2005 (Li and Staden, 1998). These ROS may initiate destructive oxidative process such as lipid peroxidation, chlorophyll bleaching, protein oxidation, and damage to nucleic acids (Scandalios, 1993).…”