Molecular Genetic Alteration of Plant Respiration (Silencing and Overexpression of Alternative Oxidase in Transgenic Tobacco)

Abstract

The alternative oxidase (AOX) of plant mitochondria is encoded by the nuclear gene Aoxl. Sense and antisense D N A constructs of Nicotiana tabacum A o x l were introduced into tobacco, and transgenic plants with both increased and decreased levels of mitochondrial AOX protein were identified. Suspension cells derived from wild-type and transgenic plants were grown in heterotrophic batch culture. Transgenic cells with increased A O X protein had an increased capacity for cyanide-resistant, salicylhydroxamic acid-sensitive respiration compared to wild-type cells, whereas transgenic cells with decreased AOX protein had a decreased capacity for such respiration. Thus, genetic alteration of the level of A O X protein was sufficient to alter the capacity for electron transport through the alternative pathway. Under our standard growth conditions, "antisense" cells with dramatically reduced levels of AOX protein had growth and respiration rates similar to the wild type. However, whereas wild-type cells were able to grow under conditions that severely suppressed cytochrome pathway activity, antisense cells could not survive this treatment. This suggests that a critical function of A O X may be to support respiration when the cytochrome pathway is impaired. The much higher level of AOX protein in "sense" cells compared to the wild type did not appreciably alter the steady-state partitioning of electrons between the cytochrome path and the alternative pathway in vivo, suggesting that this partitioning may be subject to additional regulatory factors.Higher plants have two paths of mET from UQ to O2 (Day et al., 1980;Moore and Siedow, 1991). Electron transfer through the CP is coupled to ATP synthesis. The terminal oxidase (Cyt oxidase) is inhibited by CN. Electron flow from UQ through the AP is not coupled to ATP production. The terminal oxidase is called AOX and is inhibited by SHAM. Despite a great research effort, the functional role of the AP in nonthermogenic respiratory metabolism has not been clearly defined.Studies have shown that both the capacity of the AP to support O2 consumption and the activity of the AP in con-

Decreasing Electron Flux through the Cytochrome and/or Alternative Respiratory Pathways Triggers Common and Distinct Cellular Responses Dependent on Growth Conditions

“…The development of a monoclonal antibody that recognized all AOX proteins studied to date (Elthon et al, 1989;Finnegan et al, 1999) facilitated the cloning of the gene, which allowed genetic approaches to be used to study AOX (Rhoads and McIntosh, 1991). Studies in tobacco (Nicotiana tabacum) with reduced or increased amounts of AOX (Vanlerberghe et al, 1994) showed that AOX suppresses the production of reactive oxygen species (ROS) in mitochondria (Maxwell et al, 1999;Cvetkovska and Vanlerberghe, 2012a, 2012b, 2013. Investigations with a variety of abiotic or biotic challenges revealed that AOX acts as a survival protein (Vanlerberghe et al, 2009), in that it allows plants to survive and grow better compared with plants that have reduced amounts of AOX.…”

Characterization of Transformed Arabidopsis with Altered Alternative Oxidase Levels and Analysis of Effects on Reactive Oxygen Species in Tissue

“…A third round of selection on kanamycin determined homozygous individuals (T 3 ) and these and their progeny (T 4 ) were used for further characterization. During the selection process, no morphological or fertility phenotypic differences were observed for any of the transformants, similar to other cases of transformation with AOX under CaMV 35S promoter control (Vanlerberghe et al, 1994;Millenaar, 2000;Gilliland et al, 2003).…”

“…Plants with altered AOX levels have been produced for potato (Solanum tuberosum; Hiser et al, 1996) and tobacco (Vanlerberghe et al, 1994(Vanlerberghe et al, , 1998Millenaar, 2000;Gilliland et al, 2003) using the cauliflower mosaic virus (CaMV) 35S promoter and, for Arabidopsis (Arabidopsis thaliana), using a copper-inducible promoter (Potter et al, 2001). Limited characterization of detached leaves from these transformants has been reported (Vanlerberghe et al, 1995;Potter et al, 2001) and no growth phenotypes were noted.…”

Molecular Distinction between Alternative Oxidase from Monocots and Dicots

“…Note: "Aox0" does not preclude multiple Aox-immunoreactive bands in the study referenced. Finnegan et al (1997Finnegan et al ( , 1999McCabe et al (1998);Tanudji et al (1998Tanudji et al ( , 1999; Djajanegara (2000) Vanlerberghe and McIntosh (1992a, 1992b …”