Studying Individual Plant AOX Gene Functionality in Early Growth Regulation: A New Approach
AOX1 and AOX2 genes are thought to play different physiological roles. Whereas AOX1 is typically expected to associate to stress and growth responses, AOX2 was more often found to be linked to development and housekeeping functions. However, this view is questioned by several adverse observations. For example, co-regulated expression for DcAOX1 and DcAOX2a genes was recently reported during growth induction in carrot (Daucus carota L.). Early expression peaks for both genes during the lag phase of growth coincided with a critical time point for biomass prediction, a result achieved by applying calorespirometry. The effect of both AOX family member genes cannot easily be separated. However, separate functional analysis is required in order to identify important gene-specific polymorphisms or patterns of polymorphisms for functional marker development and its use in breeding. Specifically, a methodology is missing that enables studying functional effects of individual genes or polymorphisms/polymorphic patterns on early growth regulation.
This protocol aims to provide the means for identifying plant alternative oxidase (AOX) gene variants as functional markers for early growth regulation. Prerequisite for applying this protocol is available Schizosaccharomyces pombe strains that were transformed with individual AOX genes following published protocols from Anthony Moore’s group (Albury et al., J Biol Chem 271:17062–17066, 1996; Affourtit et al., J Biol Chem 274:6212–6218, 1999). The novelty of the present protocol comes by modifying yeast cell densities in a way that allows studying critical qualitative and quantitative effects of AOX gene variants (isoenzymes or polymorphic genes) during the early phase of growth. Calorimetry is used as a novel tool to confirm differences obtained by optical density measurements in early growth regulation by metabolic phenotyping (released heat rates). This protocol enables discriminating between AOX genes that inhibit growth and AOX genes that enhance growth under comparable conditions. It also allows studying dependency of AOX gene effects on gene copy number. The protocol can also be combined with laser microdissection of individual cells from target tissues for specified breeding traits.
Key wordsEarly growth regulation AOX gene variants Gene copy number Growth performance Heterologous expression Schizosaccharomyces pombe Calorimetric analysis
At first, B.A.S. would like to appreciate the effort of the editor Jagadis Gupta Kapuganti for this special edition related to respiration and for inviting submission to make running protocols in her lab available to the public. The authors thank Isabel Velada, Hélia Cardoso, and Amaia Nogales for having established the yeast S. pombe system at the Chair’s lab in Évora by help of Mary S. Albury from the team of Anthony Moore’s group (University of Sussex, UK) and for having provided carrot AOX gene-transformed yeast variants (Velada et al. in preparation—research article). The idea of using the yeast system for studying AOX gene variants/polymorphisms had been first developed within the frame of a bilateral cooperation running between the EU Marie Curie Chair team and Anthony Moore’s group funded in Portugal by CRUP (“Concelho de Reitores das Universidades Portuguesas”) and by the British Council. Yeast transformation with carrot AOX genes was supported by Project AI/B-37/09 and Project EXCL/BEX-GMG/0038/2012 coordinated at the EU Marie Curie Chair by Hélia Cardoso. We also appreciate the consultancy of Amaia Nogales and Lee Hansen for our first steps on applying calorimetry to yeast. To Lee Hansen, B.A.S. is especially grateful for his permanently high availability as consultant at the Chair for all theoretical and practical questions related to calorimetry (see also Chapter 15 by Arnholdt-Schmitt in this book). Vinod Kumar Patil appreciates the scholarship provided by the Erasmus Mundus FUSION Project, Postdoctoral Exchange Studies (2015–2016).
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