Emerging model systems for functional genomics analysis of Crassulacean acid metabolism

Abstract

Crassulacean acid metabolism (CAM) is one of three main pathways of photosynthetic carbon dioxide fixation found in higher plants. It stands out for its ability to underpin dramatic improvements in plant water use efficiency, which in turn has led to a recent renaissance in CAM research. The current ease with which candidate CAM-associated genes and proteins can be identified through high-throughput sequencing has opened up a new horizon for the development of diverse model CAM species that are amenable to genetic manipulations. The adoption of these model CAM species is underpinning rapid advances in our understanding of the complete gene set for CAM. We highlight recent breakthroughs in the functional characterisation of CAM genes that have been achieved through transgenic approaches.

Phosphorylation of Phosphoenolpyruvate Carboxylase Is Essential for Maximal and Sustained Dark CO2 Fixation and Core Circadian Clock Operation in the Obligate Crassulacean Acid Metabolism Species Kalanchoë fedtschenkoi

“…Initial Screening and Characterization of KfPPCK1 RNAi Lines of K. fedtschenkoi As K. fedtschenkoi does not produce viable seed (Garcês et al, 2007;Hartwell et al, 2016), data are presented for primary transformants that were propagated clonally via leaf plantlets and/or stem cuttings. Primary transformants were screened initially using high-throughput leaf disc tests for starch and acidity (Cushman et al, 2008;Dever et al, 2015).…”

“…CAM into C 3 aims to improve the WUE of key C 3 crops, thereby adapting them to the increased frequency and intensity of droughts and extreme high temperatures predicted under future climate change scenarios. The further exploitation of CAM in these ways will require detailed characterization of CAM gene function, especially with respect to their daily regulation by the circadian clock Hartwell et al, 2016).…”

“…Based on our currently incomplete understanding of the molecular-genetic blueprint for CAM (Hartwell et al, 2016), C 3 species are believed to possess ancestral copies of all of the genes required for CAM (Cushman and Bohnert, 1999;Aubry et al, 2011;Ming et al, 2015). In principle, for CAM to evolve from the C 3 ancestral state in a leaf succulent CAM species such as Kalanchoë fedtschenkoi, the corresponding metabolic enzymes, metabolite transporters, and their cognate regulatory proteins must increase in abundance in leaf mesophyll cells and come under tight temporal control such that certain metabolic steps are confined to the dark period, and others are confined to the light (Cushman and Bohnert, 1999;Borland et al, 2009;Abraham et al, 2016;Brilhaus et al, 2016;Hartwell et al, 2016). However, only limited work has been reported testing this principle using a genetic approach, which represents a powerful method for in planta functional testing of candidate CAM genes when combined with detailed characterization of CAM-associated phenotypes in the resulting transgenic lines (Hartwell et al, 2016;Dever et al, 2015).…”

“…In the model obligate CAM species K. fedtschenkoi (Hartwell et al, 2016), both PPC abundance and specific activity are stable over the light/dark cycle (Nimmo et al, 1984;Dever et al, 2015), but its allosteric properties are subject to tight temporal control (Nimmo et al, 1986(Nimmo et al, , 1987. Several primary metabolites have been reported to influence the activity of PPC in vitro, including malate (allosteric inhibitor) and glucose 6-phosphate (allosteric activator) (Doncaster and Leegood, 1987;Vidal and Chollet, 1997).…”

“…PPCK remains the only well-defined, temporal/circadian control point for CAM (Hartwell, 2006(Hartwell, , 2016. To further elucidate the role of PPC phosphorylation in the temporal control of CO 2 fixation associated with CAM, and the extent to which this phospho-regulation step helps to prevent metabolic futile cycling during CAM, we generated stable transformants of K. fedtschenkoi expressing RNA interference (RNAi) constructs that targeted the degradation of endogenous KfPPCK1 transcripts.…”