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. 2019 Feb 11:10:101.
doi: 10.3389/fpls.2019.00101. eCollection 2019.

Laying the Foundation for Crassulacean Acid Metabolism (CAM) Biodesign: Expression of the C4 Metabolism Cycle Genes of CAM in Arabidopsis

Sung Don Lim  1 , Sojeong Lee  1 , Won-Gyu Choi  1 , Won Cheol Yim  1 , John C Cushman  1
Affiliations

Laying the Foundation for Crassulacean Acid Metabolism (CAM) Biodesign: Expression of the C4 Metabolism Cycle Genes of CAM in Arabidopsis

Sung Don Lim et al. Front Plant Sci. .

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.

Keywords: Arabidopsis thaliana; C4 metabolism; CAM biodesign; Mesembryanthemum crystallinum; crassulacean acid metabolism; ice plant; water-use efficiency.

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Figures

FIGURE 1
FIGURE 1
A simplified diagram of the crassulacean acid metabolism (CAM) photosynthetic pathway including key enzymes, regulatory proteins, and transporters of the C4 cycle. Key metabolites and transporters: glucose-6-phosphate (G6P), malate (MAL), phosphoenolpyruvate (PEP), beta-carbonic anhydrase (BCA), phosphoenolpyruvate carboxylase (PEPC), PEPC kinase (PPCK), NAD(P) malate dehydrogenase (NAD(P)-MDH), ribulose-1,5-bisphosphate carboxylase/oxygenase (RUBISCO), NADP-dependent malic enzyme (NADP-ME), pyruvate orthophosphate dikinase (PPDK), PPDK regulatory protein (PPDK-RP), PEP carboxykinase (PEPCK), tonoplast dicarboxylate transporter (tDT), aluminum-activated malate transporter (ALMT), vacuolar ATPase (V-ATPase), and vacuolar pyrophosphatase (V-PPiase). Red (cytosol), green (chloroplast), and purple (mitochondria) colors indicate the predicted or experimentally validated subcellular localization of each enzyme or regulatory protein fusion in this study.
FIGURE 2
FIGURE 2
Expression patterns of selected carboxylation/decarboxylation module CAM genes in M. crystallinum. To identify specific isogenes, we conducted RNA-sequencing (RNA-seq) in triplicate to define which genes were participating in CAM in the facultative CAM species M. crystallinum under well-watered (blue lines) and water-deficit stress (red lines) conditions. The averaged FPKM (fragments per kb of exon per million fragments mapped) values of three biological replicates were calculated from TMM (trimmed mean of M-values) normalized RNA-seq data. In summary, six genes of the carboxylation module (McBCA2, McPEPC1, McPPCK, McNAD-MDH1, McNAD-MDH2, and McNADP-MDH1), seven genes of the decarboxylation module (McNAD-ME1, McNAD-ME2, McNADP-ME1, McNADP-ME2, McPPDK, McPPDK-RP, and McPEPCK) were selected for initial CAM biodesign functional testing.
FIGURE 3
FIGURE 3
Subcellular localization of selected carboxylation module ice plant CAM genes expressed in A. thaliana. To identify subcellular localizations of ice plant CAM genes in A. thaliana, Agrobacterium harboring 35S::sGFP (EV control), 35S::McBCA2-sGFP, 35S::McPEPC1-sGFP, 35S::McPPCK1-sGFP, 35S::McNAD-MDH1-sGFP, 35S::McNAD-MDH2-sGFP, or 35S::McNADP-MDH1-sGFP was transformed into Arabidopsis and subcellular localization was determined by confocal microscopy. Bar = 20 μm.
FIGURE 4
FIGURE 4
Subcellular localization of selected decarboxylation module ice plant CAM genes expressed in A. thaliana. Agrobacterium harboring 35S::McNAD-ME1-sGFP, 35S::McNAD-ME2-sGFP, 35S::McNADP-ME1-sGFP, 35S::McNADP-ME2-sGFP, 35S::McPPDK-sGFP, 35S::McPPDK-RP-sGFP, or 35S::McPEPCK-sGFP was transformed into A. thaliana and subcellular localization was determined by confocal microscopy. Bar = 20 μm.
FIGURE 5
FIGURE 5
Overexpression of individual carboxylation/decarboxylation module ice plant CAM genes alters overall plant size in A. thaliana. Representative images of 4-week-old 35S::sGFP Empty Vector (EV) control, 35S::McBCA2-sGFP, 35S::McPEPC1-sGFP, 35S::McPPCK1-sGFP, 35S::McNAD-MDH1-sGFP, 35S::McNAD-MDH2-sGFP, 35S::McNADP-MDH-sGFP, 35S::McNAD-ME1-sGFP, 35S::McNAD-ME2-sGFP, 35S::McNADP-ME1-sGFP, 35S::McNADP-ME2-sGFP, 35S::McPPDK-sGFP, 35S::McPPDK-RP-sGFP, or 35S::McPEPCK-sGFP transgenic plants. Bar = 2 cm.
FIGURE 6
FIGURE 6
Overexpression of individual carboxylation/decarboxylation module ice plant CAM genes alters plant biomass in A. thaliana. T3 homozygous seeds of 35S::sGFP Empty Vector (EV) control, 35S::McBCA2-sGFP, 35S::McPEPC1-sGFP, 35S::McPPCK1-sGFP, 35S::McNAD-MDH1-sGFP, 35S::McNAD-MDH2-sGFP, 35S::McNADP-MDH1-sGFP, 35S::McNAD-ME1-sGFP, 35S::McNAD-ME2-sGFP, 35S::McNADP-ME1-sGFP, 35S::McNADP-ME2-sGFP, 35S::McPPDK-sGFP, 35S::McPPDK-RP-sGFP, and 35S::McPEPCK-sGFP were germinated and grown in soil mix under a 12-h photoperiod. 4-week-old plants were used to analyze overall plant biomass. (A) Quantification of rosette diameter (n = 10). (B) Quantification of leaf area (n = 20). (C) Quantification of leaf fresh weight (n = 20). Values represent means ± SD, ns, non-significant, ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001, One-way ANOVA with Dunnett’s multiple comparison test.
FIGURE 7
FIGURE 7
Overexpression of individual carboxylation/decarboxylation module ice plant CAM gene alters leaf stomatal conductance and malate content in A. thaliana. T3 homozygous seeds of 35S::sGFP Empty Vector (EV) control, 35S::McBCA2-sGFP, 35S::McPEPC1-sGFP, 35S::McPPCK1-sGFP, 35S::McNAD-MDH1-sGFP, 35S::McNAD-MDH2-sGFP, 35S::McNADP-MDH1-sGFP, 35S::McNAD-ME1-sGFP, 35S::McNAD-ME2-sGFP, 35S::McNADP-ME1-sGFP, 35S::McNADP-ME2-sGFP, 35S::McPPDK-sGFP, 35S::McPPDK-RP-sGFP, and 35S::McPEPCK-sGFP were germinated and grown in soil mix under a 12-h photoperiod. Four-week-old leaf was used. (A) Quantification of stomatal conductance (n = 10). (B) Quantification of malate content (n = 10). Values represent means ± SD, ns, non-significant, ∗p < 0.05 and ∗∗∗p < 0.001, One-way ANOVA with Dunnett’s multiple comparison test.
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