For many years following the elucidation of the C₄ pathway of photosynthesis in the 1960s, Kranz-type leaf anatomy was synonymous with C₄ photosynthesis. The discovery of C₄ photosynthesis in land plants that lack the Kranz anatomy in the early 2000s, revealed a much greater diversity in mechanisms of C₄ carbon fixation than previously thought (Edwards et al. 2004). Two plants, one that grows in central Asia (Borszczowia aralocaspica) and another that ranges from Anatolia eastward to Pakistan (Bienertia cycloptera), carried out complete C₄ photosynthesis in single chlorenchyma cells (Sage 2002; Edwards et al. 2004) (see textbook Figure 8.12B). Moreover, substantial evidence indicates that C₄ photosynthesis has evolved also outside the angiosperms. Some marine macroalgae and important photoautotrophic marine protists (diatoms) also perform C₄ photosynthesis in a single cell (Edwards et al. 2004).

Terrestrial Single Cell C₄ Photosynthesis

Borszczowia aralocaspica and Bienertia cycloptera (both Chenopodiaceae) can carry out C₄ photosynthesis within single chlorenchyma cells because of the uneven distribution of enzymes and chloroplasts between two regions of the same cell. This trait of the two Asian plants enables the cells to concentrate CO₂ around rubisco thus minimizing the C₂ oxidative carbon cycle (see textbook Figure 8.12B).

In Borszczowia leaves, confocal microscopy of chlorophyll autofluorescence reveals a dense layer of chloroplasts close to the vascular bundles and fewer chloroplasts near the leaf surface in contact with the atmosphere. In addition, the synthesis of the cytosolic enzyme PEPCase and the development of dimorphic chloroplasts at opposite ends of the cells accompanies the formation of chlorenchyma tissue in Borszczowia cotyledons. Chloroplasts proximal to the vascular system have well-developed grana, contain rubisco, and store starch, while those situated distally have reduced grana, contain pyruvate–phosphate dikinase and lack starch.

The assimilation of atmospheric CO₂ takes place at the chlorenchyma cells, where cytosolic PEPCase catalyzes the incorporation of HCO₃^– into phosphoenolpyruvate to form oxaloacetate, the precursor of malate. The four-carbon acid diffuses across a cytoplasmic region that is devoid of organelles to the region of the cell proximal to the vascular system. In mitochondria located at the vascular region, NAD–malic enzyme decarboxylates malate, releasing CO₂ and pyruvate and generating NADH (see textbook Table 8.4, reaction 4). The CO₂ is captured by rubisco and the Calvin–Benson cycle in chloroplasts surrounding the mitochondria. The pyruvate formed diffuses back to the region in contact with the atmosphere, where chloroplast pyruvate–phosphate dikinase converts pyruvate to phosphoenolpyruvate. Hence, these particular cells mimic intracellularly the diffusion paths of classical C₄ Kranz anatomy, positioning organelles as an adaptive response to ensure high concentrations of CO₂ around rubisco.

Marine Single Cell C₄ Photosynthesis

Diatoms are abundant unicellular algae that occur in the ocean and inland waters and account for up to 40% of the total marine primary productivity. The concentration of HCO₃^– is high in seawater (about 2 mM) but this form of inorganic carbon cannot serve as the substrate for rubisco. To overcome this limitation on the rate of carbon fixation, many eukaryotic phytoplankton species have evolved energy-dependent mechanisms for concentrating CO₂. Short-term radioactive isotope labeling experiments with the marine diatom Thalassiosira weissflogii revealed the initial incorporation of CO₂ into four-carbon acids and the subsequent transfer of carbon to 3-phosphoglycerate and sugars. Moreover, a specific inhibitor of PEPCase (i.e., 3,3-dichloro-2-dihydroxyphosphinoylmethyl-2-propenoate) inhibits photosynthesis in Thalassiosira weissflogii cells acclimated to low CO₂ (10 μM) by more than 90%. However, a subsequent increase of CO₂ to 150 μM or decrease of O₂ to 80 μM restores photosynthesis to control levels (Reinfelder et al. 2004).

Our understanding of the uptake and assimilation of inorganic carbon by diatoms advanced significantly following publication of whole genome sequences of Thalassiosira pseudonana and Phaeodactylum tricornutum. The search for enzymes involved in carbohydrate pathways in these genomes identified genes potentially involved in a C₄-like photosynthesis; i.e., PEPCase, pyruvate–phosphate dikinase, and phosphoenolpyruvate carboxykinase (Armbrust et al. 2004; Kroth et al. 2008). Up to five different isoforms of various enzymes of the C₄ cycle are believed to be distributed between plastids, mitochondria, and the cytosol. Although the subcellular localization of the initial carboxylation by PEPCase is still unclear, the functioning of the C₄ pathway in diatoms depends on the spatial separation of the decarboxylative release of CO₂ in the mitochondria from the utilization of CO₂ by rubisco in chloroplasts.