Two Cys or not two Cys, that is the question? Alternative oxidase in the thermogenic plant Nelumbo nucifera

Nicole Grant ^*, Yoshihiko Onda , Yusuke Kakizaki , Kikukatsu Ito , Jennifer Watling , and Sharon Robinson

Institute for Conservation Biology, The University of Wollongong, Australia; School of Earth and Environmental Sciences, The University of Adelaide, Australia; Cryobiofrontier Research Center, Faculty of Agriculture, Iwate University, Japan

^* Corresponding author; email: nmg944{at}uow.edu.au.

Sacred lotus (Nelumbo nucifera) regulates temperature in itsfloral chamber to 32-35°C across ambient temperatures of8-40°C with heating achieved through high alternative pathwayfluxes. In most alternative oxidase (AOX) isoforms, two cysteineresidues, Cys₁ and Cys₂, are highly conserved and play a rolein post-translational regulation of AOX. Further control occursvia interaction of reduced Cys₁ with ${alpha}$ -keto acids, such as pyruvate.Here, we report on the in vitro regulation of AOX isolated fromthermogenic receptacle tissues of sacred lotus. AOX proteinwas mostly present in the reduced form, and only a small fractioncould be oxidized with diamide. Cyanide resistant respirationin isolated mitochondria was stimulated 4-fold by succinate,but not pyruvate or glyoxylate. Insensitivity of the alternativepathway of respiration to pyruvate and the inability of AOXprotein to be oxidized by diamide suggested that AOX in thesetissues may lack Cys₁. Subsequently, we isolated 2 novel cDNAsfor AOX from thermogenic tissues of N. nucifera, designatedas NnAOX1a and NnAOX1b. Deduced amino acid sequences of bothconfirmed that Cys₁ had been replaced by serine, however Cys₂was present. This contrasts with AOXs from thermogenic Aroids,which contain both Cys₁ and Cys₂. An additional Cys was presentat position 193 in NnAOX1b. The significance of the sequencedata for regulation of the AOX protein in thermogenic sacredlotus is discussed, and compared with AOXs from other thermogenicand non-thermogenic species.