Abstract
Sacred lotus (Nelumbo nucifera) regulates temperature in its floral chamber to 32 degrees C to 35 degrees C across ambient temperatures of 8 degrees C to 40 degrees C with heating achieved through high alternative pathway fluxes. In most alternative oxidase (AOX) isoforms, two cysteine residues, Cys(1) and Cys(2), are highly conserved and play a role in posttranslational regulation of AOX. Further control occurs via interaction of reduced Cys(1) with alpha-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 Cys(1). 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 Deduced amino acid sequences of both confirmed that Cys(1) had been replaced by serine; however, Cys(2) was present. This contrasts with AOXs from thermogenic Aroids, which contain both Cys(1) and Cys(2). 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.
Highlights
2 Thermogenesis in the sacred lotus 3 The sacred lotus (Nelumbo nucifera) is a thermogenic plant that regulates the temperature of 4 its floral chamber between 32-35°C for up to 4 days (Seymour and Schultze-Motel, 1996). 5 Heating of plant tissues has been described as an adaptation to: attract insect pollinators 6 either by volatilization of scent compounds (Meeuse, 1975) or by providing a heat reward 7 (Seymour et al, 1983), protect floral parts from low temperatures (Knutson, 1974), or 8 provide the optimum temperature for floral development (Ervik and Barfod, 1999; Seymour 9 et al, 2009)
AOX protein content within the sacred lotus receptacle increases markedly prior 6 to thermogenesis, but it remains constant during heating (Grant et al, 2008), suggesting that 7 regulation of heating occurs through post-translational modification of the protein. 8 9 Post-translational regulation of AOX protein The plant alternative oxidase is a cyanide insensitive dimeric protein located in the inner mitochondrial membrane (Day and Wiskich, 1995)
Activity of sacred lotus AOX is stimulated by succinate but not pyruvate or glyoxylate Residual mitochondrial respiration rates were quite low (
Summary
AOX activity was measured using oxygen consumption of tissue, cells or isolated mitochondria in the presence or absence of AOX and COX inhibitors This method does not accurately measure activity in vivo but does indicate the ‘capacity’ of the alternative pathway (Ribas-Carbo et al, 1995; Day et al, 1996). Most AOX isoforms have two, highly conserved cysteine residues, Cys and Cys (defined by Berthold et al, 2000 and Holtzapffel et al, 2003), located near the N-terminal hydrophilic domain of the protein In these isoforms, Cys can either be reduced on both subunits of the AOX dimer or the Cys sulfhydryl groups can be oxidized to form a disulfide bridge (Rhoads et al, 1998). We further investigated AOX regulation, by 7 determining the amino acid sequence of two novel AOX genes isolated from thermogenic 8 receptacle tissue of sacred lotus. 9
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