The Birth of C2 Plants: Enhancing Photosynthetic Efficiency Through an Artificial Metabolic Cycle (Advanced)

In introductory biology courses, plant photosynthesis is commonly divided into two major stages: the light reactions and the carbon reactions, also known as the Calvin cycle. In nature, carbon fixation occurs through three major metabolic strategies: the C3 pathway, the C4 pathway, and the carbon fixation mechanism known as crassulacean acid metabolism (CAM).

Approximately 85% to 90% of angiosperms and nearly all gymnosperms rely on C3 metabolism. In this pathway, carbon dioxide diffuses into the mesophyll cells and enters the chloroplast stroma, where it is fixed directly by the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). RuBisCO catalyzes the reaction between CO₂ and ribulose-1,5-bisphosphate (RuBP), generating the three-carbon compound 3-phosphoglycerate (3PG), which then proceeds through the Calvin cycle.

Although widespread, the C3 pathway is not particularly efficient. RuBisCO possesses both carboxylase and oxygenase activities. When the oxygen concentration in the intercellular air spaces of leaves becomes relatively high, RuBisCO reacts with oxygen instead of CO₂, producing 2-phosphoglycolate (2PG). This compound is subsequently converted into glycolate and must be processed through photorespiration to prevent its accumulation and interference with metabolism. Photorespiration consumes energy and releases previously fixed carbon, resulting in substantial metabolic inefficiency.

Calvin cycle（Image source：Mike Jones， CC BY-SA 3.0 ）

To mitigate this limitation, some plants have evolved the C4 pathway. This metabolic strategy is common in members of the Poaceae and Amaranthaceae. When carbon dioxide enters the cytosol of mesophyll cells, it is first fixed by phosphoenolpyruvate carboxylase (PEPC), producing the four-carbon compound oxaloacetate. Oxaloacetate is then converted into organic acids such as malate and transported to bundle sheath cells. Inside these cells, malate is decarboxylated to generate pyruvate and CO₂, creating a locally high concentration of carbon dioxide around RuBisCO. This spatial separation greatly reduces photorespiration and enables C4 plants to maintain high photosynthetic efficiency under hot and dry conditions.

Carbon fixation in C4 plants（Image source：Derekleungtszhei， CC BY-SA 2.5 ）

Crassulacean acid metabolism (CAM) represents another adaptive strategy, found in plants such as those in the Crassulaceae, Cactaceae, Orchidaceae, and Bromeliaceae. These plants often inhabit extremely arid or saline environments. CAM plants open their stomata at night, when temperatures are lower and water loss is minimized, and absorb carbon dioxide during this period. The absorbed CO₂ is converted into organic acids such as malate and stored in vacuoles. During the day, stomata remain closed to conserve water, and the stored organic acids are decarboxylated to supply CO₂ for the Calvin cycle. This mechanism represents a temporal separation of carbon fixation and carbon assimilation.

Despite the evolution of these three strategies, researchers continue searching for ways to further improve carbon fixation efficiency in plants. One major target involves enhancing the efficiency of acetyl-coenzyme A production. Within the Calvin cycle, glyceraldehyde-3-phosphate (G3P) is produced and subsequently enters glycolysis in the cytosol, although parts of the glycolytic pathway also occur within chloroplasts. This process ultimately generates pyruvate.

Pyruvate can be converted into acetyl-coenzyme A (acetyl-CoA) in either mitochondria or chloroplasts. Acetyl-CoA is the central two-carbon precursor used in numerous biosynthetic pathways. It enters the tricarboxylic acid cycle (TCA cycle) in mitochondria and also serves as the building block for lipid synthesis, plant hormones, and many secondary metabolites. For plants, as well as for virtually all living organisms, acetyl-CoA represents one of the most fundamental metabolic intermediates. However, the Calvin-Benson-Bassham cycle primarily produces C3 compounds and is inherently inefficient at generating acetyl-CoA, because converting C3 pyruvate into the C2 molecule acetyl-CoA releases one carbon atom as CO₂, causing carbon loss during metabolism.

To address this limitation, researchers constructed an artificial dual-cycle carbon fixation system in the model plant Arabidopsis thaliana. The central component of this system is a synthetic metabolic pathway called the malyl-CoA–glycerate (McG) cycle. This engineered pathway incorporates genes encoding enzymes derived from multiple organisms:

• phosphoenolpyruvate carboxylase from Corynebacterium glutamicum

• malate thiokinase and malyl-CoA lyase from Methylococcus capsulatus

• glyoxylate carboligase from Cupriavidus necator H16

• tartronic semialdehyde reductase from Escherichia coli

• glycolate dehydrogenase from Chlamydomonas reinhardtii

McG cycle（Image source：Yu H et al. (2018)， CC BY 4.0 ）

By introducing these heterologous enzymes into the chloroplasts of Arabidopsis, the McG cycle enables two different metabolic inputs to be processed: the RuBisCO carboxylation product 3PG and the oxygenation by-product glycolate. When 3PG enters the cycle, an additional bicarbonate molecule is fixed, allowing the pathway to generate two molecules of acetyl-CoA from a single input carbon skeleton. When glycolate enters the cycle, it is converted into acetyl-CoA rather than being lost through photorespiration. This effectively recycles carbon that would otherwise be wasted.

The results of this metabolic redesign were remarkable. Arabidopsis plants containing the McG cycle exhibited biomass increases of two- to three-fold compared with wild-type plants. The number and area of rosette leaves increased substantially, and seed yield rose dramatically. Lipid accumulation in both leaves and seeds also increased significantly.

Physiological and proteomic analyses further revealed that the engineered plants possessed improved photosynthetic performance. The efficiency of photosystem II increased, and proteins involved in the photosynthetic electron transport chain were more abundant. These changes suggest that the enhanced carbon metabolism created a positive feedback loop: increased acetyl-CoA production stimulated lipid and cytokinin synthesis, which promoted growth and developmental activity, while simultaneously increasing the capacity of the photosynthetic apparatus.

An additional advantage of the McG cycle lies in its use of bicarbonate as the carbon source for phosphoenolpyruvate carboxylase. Because this enzyme uses bicarbonate rather than dissolved CO₂, it does not directly compete with RuBisCO within the chloroplast. As a result, the McG cycle can operate alongside the Calvin cycle without interfering with its function.

In nature, several ancient carbon fixation pathways—such as the reverse tricarboxylic acid cycle and the Wood–Ljungdahl pathway—can directly synthesize acetyl-CoA from CO₂ with high efficiency. However, the enzymes involved in these pathways are typically sensitive to oxygen and therefore function mainly in anaerobic organisms. As oxygen accumulated in Earth's atmosphere, most aerobic organisms evolved metabolic systems that avoid these oxygen-sensitive reactions, even though doing so sacrifices efficiency in acetyl-CoA production. The McG cycle effectively bridges these two metabolic worlds by integrating the oxygen-tolerant Calvin cycle with a synthetic pathway that efficiently generates acetyl-CoA from photorespiratory intermediates.

If this technology can be successfully transferred to crop or bioenergy plants, it could significantly increase yield, enhance lipid production, and improve carbon fixation efficiency. Such advances could have profound implications for global food production and carbon mitigation strategies. However, researchers caution that these results currently come from experiments in Arabidopsis. Whether the same metabolic engineering approach can be applied to major crops remains uncertain. Long-term expression of heterologous genes may also face risks such as gene silencing or regulatory imbalance, which will require further investigation in future studies.

Author: Shui-Ye You

Reference:

Lu KJ et al. (2025). Dual-cycle CO2 fixation enhances growth and lipid synthesis in Arabidopsis thaliana. Science.