Regulation of the Alternative Oxidase Aox1 Gene inChlamydomonas reinhardtii. Role of the Nitrogen Source on the Expression of a Reporter Gene under the Control of theAox1 Promoter

Abstract

In higher plants, various developmental and environmental conditions enhance expression of the alternative oxidase (AOX), whereas its induction in fungi is mainly dependent on cytochrome pathway restriction and triggering by reactive oxygen species. The AOX of the unicellular green alga Chlamydomonas reinhardtii is encoded by two different genes, the Aox1 gene being much more transcribed than Aox2. To analyze the transcriptional regulation of Aox1, we have fused its 1.4-kb promoter region to the promoterless arylsulfatase (Ars) reporter gene and measured ARS enzyme activities in transformants carrying the chimeric construct. We show that the Aox1 promoter is generally unresponsive to a number of known AOX inducers, including stress agents, respiratory inhibitors, and metabolites, possibly because the AOX activity is constitutively high in the alga. In contrast, the Aox1 expression is strongly dependent on the nitrogen source, being down-regulated by ammonium and stimulated by nitrate. Inactivation of nitrate reductase leads to a further increase of expression. The stimulation by nitrate also occurs at the AOX protein and respiratory levels. A deletion analysis of the Aox1 promoter region demonstrates that a short upstream segment (Ϫ253 to ϩ59 with respect to the transcription start site) is sufficient to ensure gene expression and regulation, but that distal elements are required for full gene expression. The observed pattern of AOX regulation points to the possible interaction between chloroplast and mitochondria in relation to a potential increase of photogenerated ATP when nitrate is used as a nitrogen source.Besides the cyanide-sensitive cytochrome pathway, mitochondria from higher plants, some protists, and many fungi possess an alternative pathway that is resistant to cyanide but sensitive to salicylhydroxamic acid and n-propyl gallate. Cyanide-resistant respiration is mediated by a single non-phosphorylating enzyme, the alternative oxidase (AOX), which bypasses proton-translocating complexes III and IV of the cytochrome pathway to directly transfer electrons from reduced ubiquinone to molecular oxygen. In the thermogenic spadix of Sauromatum guttatum and other Araceae, the free energy of the alternative pathway is involved in heat production during anthesis (Moore and Siedow, 1991). Although its precise function in other tissues is still not fully understood, the AOX is often considered to be a regulatory enzyme balancing carbon metabolism and electron transport. According to the energy overflow hypothesis (Lambers, 1982), shunting electrons to the alternative pathway would allow continued operation of glycolysis and tricarboxylic acid cycle when the cytochrome pathway is impaired or restricted by a high adenylate charge (for review, see Wagner and Krab, 1995;Vanlerberghe and McIntosh, 1997). The alternative pathway is also thought to prevent over-reduction of respiratory chain components that might otherwise result in the generation of harmful reactive oxygen species (for review, see Moller, 2001).Enhanc...

Characterization of the alternative oxidase of Chlamydomonas reinhardtii in response to oxidative stress and a shift in nitrogen source

“…Cells were plated on TAP NO3 medium and after about 10-14 days approximately 1% of the nit þ colonies exhibited a blue staining indicating the presence of the Aox1/Ars reporter gene and Ars activity. These finding confirmed a previous report that Aox1 is transcriptionally activated by nitrate (Baurain et al 2003). Following the preliminary characterization of some 20 independent Arsexpressing transformants, a single one was chosen for further work (clone 3).…”

“…It is doubtful, however, that these elements exist further upstream in the genome. Most C. reinhardtii regulatory sequences appear to be located within 200-300 bp from the transcription start sites (Kindle 1998), and while Baurain et al (2003) used a 1.4-kb Aox1 promoter region, it was also insensitive to antimycin A and H 2 O 2 treatment. In fact, a region within À253 to þ59 of the start site was found to be sufficient to drive the nitrate-dependent induction of Aox1 (Baurain et al 2003).…”

“…It has been suggested that AOX of the green alga C. reinhardtii is regulated in a distinctly different manner from the AOX of higher plants (Baurain et al 2003). This conclusion was based on the use of a Aox1/Ars reporter system which showed strong activation in response to a shift in nitrogen source from ammonium to nitrate -but no significant activation when cells were treated with the common inducers of Aox1 in higher plants -antimycin A and H 2 O 2 (Baurain et al 2003).…”

“…This conclusion was based on the use of a Aox1/Ars reporter system which showed strong activation in response to a shift in nitrogen source from ammonium to nitrate -but no significant activation when cells were treated with the common inducers of Aox1 in higher plants -antimycin A and H 2 O 2 (Baurain et al 2003). In the present paper, we provide evidence clearly showing that Aox1 in C. reinhardtii is induced by both antimycin A and H 2 O 2 , as well as cold-stress, but that this induction is only made apparent through the use of RNA blot analysis and not by measuring Aox1/Ars reporter gene activity.…”

Oxidative stress in fungal fermentation processes: the roles of alternative respiration

“…As with the alternative dehydrogenases, AOX does not translocate protons when electrons flow through this pathway, thus no energy is conserved but is instead again lost as heat (Moore et al 2002). This enzyme has been found in the inner membrane of mitochondria from all the higher plants, many algae, fungi, yeast and certain protozoa (Moore et al 2002 ;Baurain et al 2003 ;Juarez et al 2006). It is distinguished from the conventional cytochrome pathway by its insensitivity to cyanide, which inhibits cytochrome c oxidase (complex IV), and sensitivity to salicylhydroxamic acid (SHAM) and n-propyl gallate.…”