Two Cys or Not Two Cys? That Is the Question; Alternative Oxidase in the Thermogenic Plant Sacred Lotus    

Abstract

Sacred lotus (Nelumbo nucifera) regulates temperature in its floral chamber to 32°C to 35°C across ambient temperatures of 8°C to 40°C with heating achieved through high alternative pathway fluxes. In most alternative oxidase (AOX) isoforms, two cysteine residues, Cys1 and Cys2, are highly conserved and play a role in posttranslational regulation of AOX. Further control occurs via interaction of reduced Cys1 with α-keto acids, such as pyruvate. Here, we report on the in vitro regulation of AOX isolated from thermogenic receptacle tissues of sacred lotus. AOX protein was mostly present in the reduced form, and only a small fraction could be oxidized with diamide. Cyanide-resistant respiration in isolated mitochondria was stimulated 4-fold by succinate but not pyruvate or glyoxylate. Insensitivity of the alternative pathway of respiration to pyruvate and the inability of AOX protein to be oxidized by diamide suggested that AOX in these tissues may lack Cys1. Subsequently, we isolated two novel cDNAs for AOX from thermogenic tissues of sacred lotus, designated as NnAOX1a and NnAOX1b. Deduced amino acid sequences of both confirmed that Cys1 had been replaced by serine; however, Cys2 was present. This contrasts with AOXs from thermogenic Aroids, which contain both Cys1 and Cys2. An additional cysteine was present at position 193 in NnAOX1b. The significance of the sequence data for regulation of the AOX protein in thermogenic sacred lotus is discussed and compared with AOXs from other thermogenic and nonthermogenic species.

Analysis of Posttranslational Activation of Alternative Oxidase Isoforms

“…These other residues also may be important in relation to interactions with other organic acids. It should be noted that both groups, Holtzapffel et al (2003) and Grant et al (2009), have shown that the activating effect of succinate on oxygen consumption is not due to succinate serving as an electron donor for the electron transport chain via complex II, because AOX activation by succinate occurs even in the presence of malonate, an inhibitor of succinate dehydrogenase. Umbach et al (2002Umbach et al ( , 2006 first reported that AOX1A is activatable by 2-oxo acids at CysI and CysII.…”

“…These isoforms possess instead a Ser residue at the position of CysI. In consequence, AOX isoproteins from tomato and lotus were shown to be insensitive to 2-oxo acids but can be stimulated by the dicarboxylic acid succinate (Djajanegara et al, 1999;Ito et al, 1997;Karpova et al, 2002;Holtzapffel et al, 2003;Grant et al, 2009). Comparable to AOX isoforms that naturally possess a Ser residue at CysI, Arabidopsis AOX1A shows activation by succinate and insensitivity toward pyruvate or glyoxylate after substitution of CysI by Ser (Djajanegara et al, 1999).…”

“…S1). These AOX isoforms were shown to be insensitive to 2-oxo acids but stimulated by succinate (Ito et al, 1997;Karpova et al, 2002;Holtzapffel et al, 2003;Grant et al, 2009). Consequently, various single-, double-, and triple-substituted derivatives of AOX1A, AOX1C, and AOX1D were systematically analyzed for their sensitivity toward succinate (Fig.…”

Identification of a Gene for Pyruvate-Insensitive Mitochondrial Alternative Oxidase Expressed in the Thermogenic Appendices in Arum maculatum

“…These thermogenic plants can be categorized into two groups: namely, a group including skunk cabbage (Symplocarpus renifolius) and sacred lotus (Nelumbo nucifera), which have been shown to maintain their flower temperatures over several days by active thermoregulation (Ito et al, 2003b), and a group containing A. maculatum and S. guttatum (Crichton et al, 2005), both of which show transient (4-6 h) and rather uncontrolled heat production. It has also been shown that the sacred lotus, a dicot, expresses at least two AOX proteins lacking a conserved CysI, termed NnAOX1a and NnAOX1b, both of which have been suggested to be stimulated by succinate (Grant et al, 2009). Moreover, it has been demonstrated that skunk cabbage expresses a pyruvate-sensitive ENV type of AOX, SrAOX, in this thermogenic spadix (Onda et al, 2007).…”

In the heat of the night – alternative pathway respiration drives thermogenesis in Philodendron bipinnatifidum

“…Activation of AOX is controlled, in part, by the redox status of the protein, which is regulated via the formation of disulfide bonds between conserved cysteine residues (Rhoads et al, 1998). At least one isoform of AOX from P. bipinnatifidum contains the regulatory cysteines (Ito & Seymour, 2005;Grant et al, 2009); however, c. 40% of the protein resists oxidation by diamide (Grant, 2010), suggesting it may lack this redox control. The activity of the reduced protein can be further moderated by effectors such as a-keto acids (e.g.…”