The relationships between heat production, alternative oxidase (AOX) pathway flux, AOX protein, and carbohydrates during floral development in Nelumbo nucifera (Gaertn.) were investigated. Three distinct physiological phases were identified: pre-thermogenic, thermogenic, and post-thermogenic. The shift to thermogenic activity was associated with a rapid, 10-fold increase in AOX protein. Similarly, a rapid decrease in AOX protein occurred post-thermogenesis. This synchronicity between AOX protein and thermogenic activity contrasts with other thermogenic plants where AOX protein increases some days prior to heating. AOX protein in thermogenic receptacles was significantly higher than in post-thermogenic and leaf tissues. Stable oxygen isotope measurements confirmed that the increased respiratory flux supporting thermogenesis was largely via the AOX, with little or no contribution from the cytochrome oxidase pathway. During the thermogenic phase, no significant relationship was found between AOX protein content and either heating or AOX flux, suggesting that regulation is likely to be post-translational. Further, no evidence of substrate limitation was found; starch accumulated during the early stages of floral development, peaking in thermogenic receptacles, before declining by 89% in post-thermogenic receptacles. Whilst coarse regulation of AOX flux occurs via protein synthesis, the ability to thermoregulate probably involves precise regulation of AOX protein, most probably by effectors such as alpha-keto acids.

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.

Summary• Philodendron bipinnatifidum inflorescences heat up to 42°C and thermoregulate. We investigated whether they generate heat via the cytochrome oxidase pathway uncoupled by uncoupling proteins (pUCPs), or the alternative oxidase (AOX).• Contribution of AOX and pUCPs to heating in fertile (FM) and sterile (SM) male florets was determined using a combination of oxygen isotope discrimination, protein and substrate analyses.• Both FM and SM florets thermoregulated independently for up to 30 h ex planta. In both floret types, AOX contributed > 90% of respiratory flux during peak heating. The AOX protein increased fivefold with the onset of thermogenesis in both floret types, whereas pUCP remained low throughout development. These data indicate that AOX is primarily responsible for heating, despite FM and SM florets potentially using different substrates, carbohydrates or lipids, respectively. Measurements of discrimination between O 2 isotopes in strongly respiring SM florets were affected by diffusion; however, this diffusional limitation was largely overcome using elevated O 2 .• The first in vivo respiratory flux measurements in an arum show AOX contributes the bulk of heating in P. bipinnatifidum. Fine-scale regulation of AOX activity is post-translational. We also demonstrate that elevated O 2 can aid measurement of respiratory pathway fluxes in dense tissues.