Abstract
Crassulacean acid metabolism (CAM) is a specialized mode of photosynthesis that exploits a temporal CO2 pump with nocturnal CO2 uptake and concentration to reduce photorespiration, improve water-use efficiency (WUE), and optimize the adaptability of plants to hotter and drier climates. Introducing the CAM photosynthetic machinery into C3 (or C4) photosynthesis plants (CAM Biodesign) represents a potentially breakthrough strategy for improving WUE while maintaining high productivity. To optimize the success of CAM Biodesign approaches, the functional analysis of individual C4 metabolism cycle genes is necessary to identify the essential genes for robust CAM pathway introduction. Here, we isolated and analyzed the subcellular localizations of 13 enzymes and regulatory proteins of the C4 metabolism cycle of CAM from the common ice plant in stably transformed Arabidopsis thaliana. Six components of the carboxylation module were analyzed including beta-carbonic anhydrase (McBCA2), phosphoenolpyruvate carboxylase (McPEPC1), phosphoenolpyruvate carboxylase kinase (McPPCK1), NAD-dependent malate dehydrogenase (McNAD-MDH1, McNAD-MDH2), and NADP-dependent malate dehydrogenase (McNADP-MDH1). In addition, seven components of the decarboxylation module were analyzed including NAD-dependent malic enzyme (McNAD-ME1, McNAD-ME2), NADP-dependent malic enzyme (McNADP-ME1, NADP-ME2), pyruvate, orthophosphate dikinase (McPPDK), pyruvate, orthophosphate dikinase-regulatory protein (McPPDK-RP), and phosphoenolpyruvate carboxykinase (McPEPCK). Ectopic overexpression of most C4-metabolism cycle components resulted in increased rosette diameter, leaf area, and leaf fresh weight of A. thaliana except for McNADP-MDH1, McPPDK-RP, and McPEPCK. Overexpression of most carboxylation module components resulted in increased stomatal conductance and dawn/dusk titratable acidity (TA) as an indirect measure of organic acid (mainly malate) accumulation in A. thaliana. In contrast, overexpression of the decarboxylating malic enzymes reduced stomatal conductance and TA. This comprehensive study provides fundamental insights into the relative functional contributions of each of the individual components of the core C4-metabolism cycle of CAM and represents a critical first step in laying the foundation for CAM Biodesign.
Highlights
Crassulacean acid metabolism (CAM) is a temporally controlled, inorganic carbon-concentrating mechanism that improves water-use efficiency (WUE) by shifting all or part of CO2 uptake from the day to the night when air:leaf water vapor pressure deficits are lower compared with the day (Griffiths, 1989)
The C4 acids accumulated overnight are subsequently decarboxylated during the day by either NAD(P)-malic enzyme (ME) to release pyruvate and CO2 and pyruvate orthophosphate dikinase (PPDK) to regenerate the pyruvate to PEP or NAD(P)-malate dehydrogenase (MDH) and PEP carboxykinase (PEPCK) to release CO2 and regenerate PEP, depending on the species (Winter, 1985; Christopher and Holtum, 1996; Kondo et al, 2000)
CAM is characterized by a core C4-metabolism carboxylation module of three major enzymes and a regulatory protein kinase leading to nocturnal CO2 uptake and fixation leading to the formation of malate, which is transported and stored in the vacuole overnight as malic acid (Figure 1)
Summary
Crassulacean acid metabolism (CAM) is a temporally controlled, inorganic carbon-concentrating mechanism that improves water-use efficiency (WUE) by shifting all or part of CO2 uptake from the day to the night when air:leaf water vapor pressure deficits are lower compared with the day (Griffiths, 1989). Detailed knowledge of the functions of the enzymatic, transport, and regulatory components is required prior to engaging in CAM Biodesign efforts Such information is critical for creating and refining metabolic flux balance analysis models of CAM (Cheung et al, 2014) and performing computational analyses of the productivity potential of CAM and engineered CAM (Shameer et al, 2018). To this end, facultative CAM plants provide a useful means of determining precisely which gene family members are recruited to function in CAM (Cushman et al, 2008; Winter and Holtum, 2014; Hartwell et al, 2016). Proteomic analyses have revealed differential protein abundance changes of CAMrelated enzymes in various cell types and subcellular fractions triggered by salinity stress treatment (Barkla et al, 2012, 2016; Cosentino et al, 2013)
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