A novel single-cell NAD-ME C₄ subtype integrated with CAM and bicarbonate use in an aquatic plant

Many plants maximize photosynthesis by using a CO₂-concentrating mechanism (CCM). Based on physiology, the freshwater plant Ottelia alismoides has three CCMs: C₄ metabolism (NAD-malic enzyme (NAD-ME) subtype) and bicarbonate-use during the day plus crassulacean acid metabolism (CAM) at night and lacks Kranz anatomy. Here, we combined a range of techniques including analysis of enzyme activity and location, transcriptomics, proteomics and ¹³C labelling in plants grown at low and high concentrations of CO₂ to investigate how these CCMs interact and can be integrated without Kranz anatomy. We showed that, unlike canonical NAD-ME subtypes, malate is the first stable compound, produced by a cytosolic malate dehydrogenase, rather than aspartate produced by aspartate aminotransferase. CAM depends on the nocturnal synthesis and transport of malic acid into the vacuole involving a vacuolar-ATPase and a tonoplast dicarboxylate transporter that are highly expressed at night. These results show that C₄ and CAM are compatible within a single cell, thanks to temporal regulation and expression of different isoforms of key enzymes and transporters. They contribute to the growing appreciation of the diversity of CCMs and how different processes can co-occur and be coordinated. This study presents a model that could facilitate future plant engineering.