For decades, plant scientists faced a fascinating mystery. Common purslane (Portulaca oleracea), a widespread weed, was known to perform two powerful types of photosynthesis that seemed fundamentally incompatible.
Like corn or sugarcane, it uses efficient C4 photosynthesis when water is plentiful, achieving high growth rates. Remarkably, when drought strikes, it switches gears and activates Crassulacean Acid Metabolism (CAM), a water-saving trick used by cacti.
The big question was how? Scientists believed C4 and CAM couldn’t work together in the same plant cells because they require different setups in space and time. C4 splits the work between two cell types (mesophyll and bundle sheath) spatially, while CAM splits the work between night and day within the same cell temporally.
Combining them risked metabolic chaos. However, groundbreaking research using advanced techniques has now cracked this puzzle wide open, revealing purslane’s unique and highly efficient integrated system.
Advanced Gene Mapping Reveals Secrets
To uncover purslane’s secret, researchers employed sophisticated tools. First, they confirmed the plant truly uses CAM under drought by measuring acid buildup overnight, a key CAM signature. Well-watered plants showed no significant night-time acid increase.
However, after just seven days without water, acid levels at dawn were dramatically higher than at dusk, proving CAM was active.
Next, the team used laser precision. They carefully isolated pure mesophyll cells and bundle sheath cells from leaves under different conditions (well-watered/drought, dawn/dusk) and sequenced their active genes (RNA).
This generated massive data – over a billion genetic reads analyzed across 28 samples. Furthermore, they used spatial transcriptomics (Visium technology), which acts like a molecular GPS.
This technique maps gene activity directly onto images of thin leaf slices, showing exactly where specific genes are turned on – pinpointing mesophyll, bundle sheath, or water storage cells.
Finally, they built complex computer models (Flux Balance Analysis) to simulate the flow of chemicals through all possible pathways in the leaf cells, predicting the most efficient way to produce sugars under stress.
C4 CAM Integration in Single Cells
The results painted a clear and revolutionary picture. Contrary to old assumptions, both C4 and CAM activities primarily happen within the same mesophyll cells, but cleverly separated in time using different genetic switches.
Under drought, a specific CAM version of the enzyme PEP carboxylase (called PPC-1E1c) skyrockets in activity only at night within mesophyll cells (a massive 5.6-fold increase compared to daytime levels).
Simultaneously, the standard C4 version of PEPC (PPC-1E1a’) remains active in these same cells during the day. Crucially, the water storage cells showed almost no activity of these key genes, debunking the idea that CAM was outsourced.
The night shift CAM activity produces malic acid, which gets stored inside the mesophyll cell vacuoles. Here’s the masterstroke: during the day, this CAM-produced malate isn’t broken down in the mesophyll.
Instead, it gets transported to the bundle sheath cells. Once there, it seamlessly enters the plant’s existing C4 processing line. The bundle sheath cells, specialized for this task, break down the malate (using enzymes like NAD-ME-2E) releasing CO2 right where the Rubisco enzyme works most efficiently.
Essentially, CAM acts as a highly efficient night-time carbon capture team in the mesophyll, feeding its product directly into the high-capacity daytime C4 factory in the bundle sheath. The computer models confirmed this integration is optimal, showing strong correlations (R² up to 0.93) between predicted enzyme activity and actual gene expression data.
High Growth Meets Extreme Drought Tolerance
The power of this integrated C4+CAM system isn’t just theoretical; the study quantified its remarkable advantages. Purslane maintains impressive C4-like photosynthesis rates of about 23 μmol of CO2 captured per square meter per second when water is abundant.
More importantly, under drought stress simulated by the model (45% reduction in CO2 intake due to closed stomata), the integrated system showed incredible resilience. Phloem output, which carries vital sugars away from the leaves, dropped by only 8% compared to well-watered conditions.
This minimal loss starkly contrasts with a predicted 52% crash in sugar output if the plant relied only on C4 without the CAM component under the same drought stress.
The model revealed significant metabolic fluxes driving this resilience: substantial nocturnal malate storage in mesophyll vacuoles (47.2 mmol per gram of dry weight per day) and a significant daytime flow of this CAM-produced malate from mesophyll to bundle sheath (40.8 mmol gDW⁻¹ day⁻¹) for processing.
This efficiency stems from leveraging the existing, powerful C4 machinery in the bundle sheath to handle the carbon captured by the drought-induced CAM system, maximizing output with minimal extra cost. Consequently, purslane breaks a fundamental rule in plant biology, achieving both high productivity and exceptional drought tolerance.
Blueprint for Climate-Resilient Crop Engineering
Understanding purslane’s quantifiable success opens exciting doors for agriculture, especially as climate change intensifies droughts. While engineering full C4 photosynthesis into rice or full CAM into standard crops remains complex, this research suggests a more targeted and potentially achievable strategy: adding a facultative, drought-responsive CAM module into the mesophyll cells of existing C4 crops like corn (maize), sorghum, or sugarcane.
The key insight is that these crops already possess the efficient bundle sheath “processing plant” perfectly capable of handling malate. Purslane proves that CAM-produced malate can directly feed into this existing C4 decarboxylation pathway.
Therefore, the main engineering focus would be on giving these crops the mesophyll-based night-shift carbon capture ability seen in purslane. This involves introducing genes for drought-induced, night-active versions of enzymes like the specific PEPC isoform (PPC-1E1c, which showed a 5.6 log2FC increase), relevant carbonic anhydrase, malate dehydrogenase, and regulators like PEPC kinase, along with enhancing vacuolar storage capacity for malate.
Encouragingly, purslane’s data indicates this integration works within standard C4 leaf anatomy without needing entirely new cell types or massive restructuring. Purslane itself, with its short life cycle and available genome, now serves as an excellent model system for testing these concepts.
The quantified benefit – only an 8% productivity loss under severe drought versus 52% without CAM – provides a compelling target for future crop improvement, offering a realistic pathway to developing crops that can maintain yields on a hotter, drier planet by mimicking nature’s ingenious solution found in a common weed.
Key Terms and Concepts
What is C4 Photosynthesis: A special plant process that efficiently captures carbon dioxide (CO2) in one cell type (mesophyll) and concentrates it for sugar production in another cell type (bundle sheath), minimizing wasteful photorespiration. It’s crucial for high growth rates in hot, sunny environments. Examples include maize and sugarcane. It involves initial CO2 fixation by PEPC into a 4-carbon acid (like malate).
What is CAM (Crassulacean Acid Metabolism): A water-saving adaptation where plants open stomata at night to take in CO2, fixing it into malic acid stored in vacuoles. During the day, stomata close, and the stored acid breaks down, releasing CO2 for photosynthesis internally. It’s vital for survival in dry habitats. Examples are cacti and pineapple. Malate accumulation and decarboxylation are key steps.
What is Mesophyll Cell: The main photosynthetic tissue in plant leaves, typically packed with chloroplasts. In C4 plants like Portulaca, it performs initial CO2 fixation using PEPC, both for the standard C4 cycle and the induced CAM cycle. Its function is critical for capturing carbon. These cells are located between the leaf veins.
What is Bundle Sheath Cell: Cells tightly surrounding the leaf veins. In C4 plants, they receive 4-carbon acids (like malate) from mesophyll cells, decarboxylate them to release concentrated CO2, and use RuBisCO to fix this CO2 into sugars via the Calvin cycle. They are essential for the spatial separation in C4 efficiency.
What is PEPC (Phosphoenolpyruvate Carboxylase): A key enzyme that grabs CO2 (as bicarbonate) and attaches it to a 3-carbon molecule (PEP) to make a 4-carbon acid (oxaloacetate). It initiates carbon fixation in both C4 and CAM photosynthesis. Its activity is central to concentrating CO2. Formula: PEP + HCO3- → Oxaloacetate + Pi.
What is RuBisCO (Ribulose-1,5-bisphosphate carboxylase/oxygenase): The main enzyme of the Calvin cycle that fixes CO2 into organic molecules to make sugars. In C4 plants, it works almost exclusively in the bundle sheath cells where CO2 concentration is high, reducing wasteful oxygenation (photorespiration). It’s fundamental to life.
What is Malate: A 4-carbon organic acid. In C4 plants, it’s a major molecule transporting CO2 from mesophyll to bundle sheath cells. In CAM plants, it’s the primary form storing CO2 (as malic acid) in vacuoles overnight. Portulaca uses malate for both pathways. Formula: C4H6O5.
What is Decarboxylation: The chemical reaction where a carboxyl group (-COOH) is removed from a molecule, releasing CO2. In C4 photosynthesis, this happens in bundle sheath cells (e.g., via NAD-ME), releasing concentrated CO2 for RuBisCO. In CAM, daytime decarboxylation of malate releases stored CO2 for fixation.
What is Calvin Cycle: The set of light-independent reactions in photosynthesis where RuBisCO fixes CO2 into a 3-carbon sugar (3-phosphoglycerate), eventually building glucose and other carbohydrates using energy (ATP, NADPH) from the light reactions. It occurs in chloroplasts. This cycle produces the plant’s food.
What is Flux Balance Analysis (FBA): A computational method using mathematical models to predict the flow (flux) of metabolites through a metabolic network, aiming to maximize a goal (like growth or sugar output) under given constraints. The study used FBA to model and confirm the efficiency of the integrated C4+CAM system in Portulaca.
What is Gene Expression: The process where information in a gene is used to make a functional product, usually a protein, involving transcription (making mRNA) and translation. The study measured expression levels (via mRNA abundance) of C4 and CAM genes in different cells and conditions to locate the pathways.
What is Transcriptomics: The study of all the RNA molecules (transcriptome), especially messenger RNA (mRNA), in a cell or tissue, revealing which genes are actively being expressed. Researchers used transcriptomics (via RNA sequencing) to identify which C4 and CAM genes were active in mesophyll and bundle sheath cells.
What is Laser Capture Microdissection (LCM): A technique using a laser to precisely cut out and collect specific cells (like mesophyll or bundle sheath) from a tissue section under a microscope. This allowed the researchers to isolate these cell types and analyze their specific gene expression profiles separately.
What is Spatial Transcriptomics (e.g., Visium): A technology that maps where genes are expressed within a tissue section by capturing mRNA directly on a slide with position-specific barcodes. The study used Visium to visualize that C4 and CAM genes were co-expressed in the mesophyll tissue, confirming integration.
What is Facultative CAM: The ability of a plant to switch on CAM photosynthesis only under stressful conditions (like drought) while normally using C3 or C4 metabolism. Portulaca oleracea is a C4 plant that uses facultative CAM when water-stressed, combining high productivity with drought tolerance.
What is Kranz Anatomy: The specialized leaf structure in C4 plants where bundle sheath cells, rich in chloroplasts, form a ring (“wreath” = Kranz) around the veins, surrounded by mesophyll cells. This anatomy enables the spatial separation of initial CO2 fixation (mesophyll) and the Calvin cycle (bundle sheath).
What is Photorespiration: A wasteful process where RuBisCO binds oxygen (O2) instead of CO2, especially when CO2 is low and O2 is high, consuming energy and releasing fixed carbon. C4 and CAM photosynthesis evolved primarily to suppress photorespiration by concentrating CO2 around RuBisCO.
What is Water Use Efficiency (WUE): The ratio of carbon fixed (photosynthesis) to water lost (transpiration). CAM dramatically improves WUE by opening stomata only at night when humidity is higher and evaporation is lower, minimizing water loss while still fixing CO2. This is crucial in arid environments.
What is Carbon Concentration Mechanism (CCM): A biochemical or structural adaptation that increases CO2 concentration around RuBisCO to minimize photorespiration. Both C4 (spatial concentration via cell separation) and CAM (temporal concentration via night fixation and day release) are types of CCMs.
What is Ortholog: Genes in different species that evolved from a common ancestral gene and typically retain the same function. The study identified specific orthologs of genes like PEPC that were specialized for either C4 (PPC-1E1a’) or CAM (PPC-1E1c) function in Portulaca.
What is Paralog: Genes within the same species that arose from gene duplication events and may evolve new or specialized functions. Portulaca has paralogs of key enzymes (e.g., PEPC genes), where one copy is used for C4 photosynthesis and another for CAM, allowing pathway separation.
What is Differential Gene Expression (DE): The phenomenon where genes show significant differences in their expression levels (amount of mRNA produced) under different conditions (e.g., drought vs. watered) or in different cell types (e.g., mesophyll vs. bundle sheath). DE analysis identified cell-specific C4/CAM genes.
What is Metabolic Flux: The rate at which metabolites flow through specific biochemical pathways in a cell. FBA predicted the fluxes (e.g., how much malate moves from mesophyll to bundle sheath) in the integrated C4+CAM system, showing how the pathways interconnect efficiently.
What is Titratable Acidity: A measurement of the total acid concentration in a solution, determined by how much base (like NaOH) is needed to neutralize it to pH 7. The study used diel changes in leaf titratable acidity to confirm CAM induction (night acid accumulation) under drought.
What is Specific Leaf Area (SLA): The ratio of leaf area to its dry mass (cm²/g or m²/kg). It relates to leaf thickness and resource investment. The study used the SLA of Portulaca oleracea (600 cm²/g) to convert model-predicted metabolic fluxes (per gram dry weight) to leaf area-based CO2 uptake rates.
Reference:
Moreno-Villena, J. J., Zhou, H., Gilman, I. S., Tausta, S. L., Cheung, C. M., & Edwards, E. J. (2022). Spatial resolution of an integrated C4+ CAM photosynthetic metabolism. Science Advances, 8(31), eabn2349. https://doi.org/10.1126/sciadv.abn2349
