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

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 Deduced amino acid sequences of both confirmed that Cys₁ had been replaced by serine; however, Cys₂ was present.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.