Identification of superoxide production by Arabidopsis thaliana aldehyde oxidases AAO1 and AAO3

Abstract

Plant aldehyde oxidases (AOs) have gained great attention during the last years as they catalyze the last step in the biosynthesis of the phytohormone abscisic acid by oxidation of abscisic aldehyde. Furthermore, oxidation of indole-3-acetaldehyde by AOs is likely to represent one route to produce another phytohormone, indole-3-acetic acid, and thus, AOs play important roles in many aspects of plant growth and development. In the present work we demonstrate that heterologously expressed AAO1 and AAO3, two prominent members of the AO family from Arabidopsis thaliana, do not only generate hydrogen peroxide but also superoxide anions by transferring aldehyde-derived electrons to molecular oxygen. In support of this, superoxide production has also been found for native AO proteins in Arabidopsis leaf extracts. In addition to their aldehyde oxidation activity, AAO1 and AAO3 were found to exhibit NADH oxidase activity, which likewise is associated with the production of superoxide anions. According to these results and due to the fact that molecular oxygen is the only known physiological electron acceptor of AOs, the production of hydrogen peroxide and/or superoxide has to be considered in any physiological condition in which aldehydes or NADH serve as substrate for AOs. In this respect, conditions such as natural senescence and stress-induced stomatal movement, which both require simultaneously elevated levels of abscisic acid and hydrogen peroxide/superoxide, are likely to benefit from AOs in two ways, namely by formation of abscisic acid and by concomitant formation of reactive oxygen species.

The Biosynthetic Pathway of Indole-3-Carbaldehyde and Indole-3-Carboxylic Acid Derivatives in Arabidopsis

“…Possibly, cyanide released by CYP71B6 and other enzymes catalyzing the same reaction is recycled efficiently into central metabolism. As both cytochrome P450s as well as AAO1 (Zarepour et al, 2012) are inhibited by cyanide, rapid removal of this by-product is necessary to prevent shutdown of the biosynthetic pathway. Recently, it was demonstrated that low amounts of cyanide (approximately 0.08 nmol g 21 fresh weight) accumulate in response to Botrytis cinerea infection (García et al, 2013).…”

“…ARABIDOPSIS ALDEHYDE OXIDASE3 (AAO3) catalyzes the last step in abscisic acid biosynthesis , and AAO4 is responsible for benzaldehyde oxidation in siliques (Ibdah et al, 2009). AAO1 and AAO2 as homodimers and heterodimers have a rather broad substrate specificity in vitro (Akaba et al, 1999;Koiwai et al, 2000), and AAO1 was recently shown to catalyze the NADH-dependent production of superoxide anions (Zarepour et al, 2012). It was proposed that aldehyde oxidases are involved in auxin biosynthesis (Seo et al, 1998), and one substrate of the AAO1 homomer is indole-3-acetaldehyde (IAAld), which is oxidized to indole-3-acetic acid (IAA) with an apparent K m value of 39 mM .…”

Aldehyde Oxidase 4 Plays a Critical Role in Delaying Silique Senescence by Catalyzing Aldehyde Detoxification

“…S8 (Zarepour et al, 2012), we wished to examine whether AAO4 was capable of the same. Additionally, the suitability of various kinds of aldehydes for the generation of ROS by AAO4 was examined, employing aliphatic and aromatic aldehydes after protein fractionation using in-gel assays for O 2 2 -and H 2 O 2 -dependent activity band generation (Sagi and Fluhr, 2001;Yesbergenova et al, 2005).…”

“…Additionally, Ibdah et al (2009), in their AAO4 activity assay, employed NAD + as a cosubstrate together with the aldehyde substrate. However, the use of NAD + by plants, or for that matter by any eukaryotic AO, is not known (Nishino and Nishino, 1989;Terao et al, 1998;Garattini et al, 2009;Kundu et al, 2012;Zarepour et al, 2012;Li et al, 2014). Moreover, Coelho et al (2012) recently demonstrated that the lack of residues leading to a flexible loop around the FAD domain in mammalian AO crystal structure can account for the fact that AOs are pure oxidases and cannot use NAD + as the final acceptor of reducing equivalents.…”

“…One explanation might be that simultaneous transfer takes place of one or two electrons from the oxidized aldehyde via the FAD domain of the enzyme to molecular oxygen, to generate, preferably, H 2 O 2 in the case of two-electron transfer and O 2 2 in the case of one-electron transfer. Another possibility is that, in AAO4, as in sulfite oxidase (which, like AAO4, belongs to the family of the molybdoenzymes), O 2 2 is generated during sulfite oxidation by the transfer of one electron to molecular oxygen but that H 2 O 2 is detected as a result of the subsequent spontaneous oxidation of O 2 2 (Hänsch et al, 2006;Byrne et al, 2009;Zarepour et al, 2012). In this second scenario, the H 2 O 2 generated by AAO4 would be the result of spontaneous dismutation of the generated O 2 2 .…”