Stomatal Conductance and Mesophyll Limitations in C3-C4 Intermediate Species Under Elevated CO2 Conditions

Introduction

The ongoing increase in atmospheric carbon dioxide concentrations represents one of the most significant environmental changes affecting plant productivity and ecosystem function. Understanding how plants respond to elevated CO2 is crucial for predicting agricultural productivity and natural ecosystem dynamics under future climate scenarios. Among the various plant functional types, C3-C4 intermediate species occupy a particularly interesting position in this context, as they possess partially developed CO2-concentrating mechanisms that may confer unique adaptive advantages under changing atmospheric conditions. C3-C4 intermediate photosynthesis represents an evolutionary transition state between the ancestral C3 pathway and the more derived C4 pathway. These intermediate species typically exhibit reduced CO2 compensation points compared to C3 plants, achieved through the refixation of photorespired CO2 in bundle sheath cells. This partial CO2-concentrating mechanism results from altered anatomical features, including enlarged bundle sheath cells with increased organelle content, and modified biochemical pathways involving the differential localization of glycine decarboxylase to bundle sheath mitochondria. The genus Flaveria has emerged as a model system for investigating C3-C4 intermediacy, containing species spanning the complete range from full C3 to full C4 photosynthesis. in the release of photorespired CO2 in a cellular compartment partially isolated from the atmosphere. The magnitude of this CO2-concentrating effect depends on bundle sheath anatomy, particularly the surface area to volume ratio and the presence or absence of suberized lamellae that may restrict gas diffusion. Stomatal conductance can be quantified from gas exchange measurements as the ratio of transpiration rate to leaf-to-air vapor pressure deficit, corrected for boundary layer effects.

Mesophyll conductance is typically estimated using concurrent measurements of gas exchange and carbon isotope discrimination or chlorophyll fluorescence, based on the relationship between these measurements and the CO2 concentration drawdown from substomatal cavities to chloroplasts.

Material and Methods Material

Three C3-C4 intermediate species were selected for this research: Flaveria floridana, Flaveria ramosissima, and Mollugo verticillata. Seeds were obtained from established germplasm collections and germinated in controlled environment chambers maintained at 25°C with a 14-hour photoperiod. Seedlings were transplanted to 5-L pots containing a peat-vermiculite-perlite mixture (2:1:1 by volume) and grown for eight weeks under ambient atmospheric conditions before CO2 treatment initiation.

Plants were divided into two treatment groups: ambient CO2 (400±20 ppm) and elevated CO2 (800±30 ppm). CO2 concentrations were maintained using automated mass flow controllers that supplied CO2-enriched or CO2-depleted air to sealed growth chambers. Environmental conditions during the treatment period included day/night temperatures of 28/22°C, relative humidity of 60±5%, and photosynthetically active radiation of 600 μmol m⁻² s⁻¹ provided by metal halide lamps with supplemental far-red LEDs.

Methods

Gas exchange measurements were conducted after six weeks of CO2 treatment using a portable photosynthesis system equipped with a leaf chamber fluorometer. Measurements were made on fully expanded leaves between 09:00 and 14:00 hours to minimize diurnal variation. Leaf chamber conditions were standardized at 25°C, 60% relative humidity, and 1500 μmol m⁻² s⁻¹ photosynthetically active radiation. CO2 response curves were generated by varying the reference CO2 concentration from 50 to 2000 ppm in stepwise increments.

Stomatal conductance was calculated from transpiration measurements using the manufacturer software. Mesophyll conductance was estimated using the variable J method, which combines gas exchange and chlorophyll fluorescence measurements. This method assumes that under nonphotorespiratory conditions, the electron transport rate calculated from gas exchange equals that calculated from fluorescence measurements, with the difference under photorespiratory conditions reflecting the CO2 drawdown across the mesophyll.

Carbon isotope discrimination was measured on leaf samples collected immediately after gas exchange measurements. Samples were oven-dried at 60°C for 72 hours, ground to fine powder, and analyzed using an isotope ratio mass spectrometer coupled to an elemental analyzer.

Discrimination was calculated relative to the carbon isotope composition of the growth chamber CO2. Limitation analysis was performed following established protocols that partition total photosynthetic limitation into stomatal, mesophyll, and biochemical components. The relative limitation imposed by each component was calculated as the proportional reduction in assimilation rate that would occur if that component were eliminated while others remained unchanged.

Physiological Measurements

Chlorophyll fluorescence parameters were measured concurrently with gas exchange using the integrated fluorometer in the leaf chamber. Maximum fluorescence was determined after a saturating light pulse of 8000 μmol m⁻² s⁻¹ for 0.8 seconds. Steady-state fluorescence was recorded under actinic light, and the effective quantum yield of photosystem II was calculated. Electron transport rates were estimated from fluorescence parameters assuming an even distribution of absorbed light between photosystems. Leaf water potential was measured predawn and midday using a pressure chamber technique. Leaves were excised at the petiole, immediately sealed in humidified plastic bags, and transported to the laboratory for measurement within 30 minutes of collection. Hydraulic conductance was calculated from the relationship between transpiration rate and the soil-to-leaf water potential gradient. Leaf nitrogen content was determined by combustion analysis using an elemental analyzer. Samples were dried at 60°C for 72 hours before analysis. Specific leaf area was calculated as the ratio of leaf area (determined by digital image analysis) to dry mass.

Results

Table Mesophyll conductance exhibited species-specific responses to elevated CO2 (Table 2). F. floridana showed a substantial decrease of 31.4%, while F. ramosissima maintained relatively stable mesophyll conductance with only a 4.8% reduction. The partitioning of diffusional limitations shifted under elevated CO2, with stomatal limitations decreasing in relative importance while mesophyll limitations increased proportionally. Electron transport rates increased significantly under elevated CO2 across all species, with enhancements ranging from 15.8% in M. verticillata to 24.3% in F. ramosissima (Table 3). The maximum carboxylation rate of Rubisco showed modest increases under elevated CO2, while the maximum electron transport rate remained relatively unchanged, suggesting potential limitations in RuBP regeneration capacity.

Comprehensive Interpretation

The differential responses of stomatal and mesophyll conductance to elevated CO2 among the C3-C4 intermediate species investigated reveal distinct physiological strategies that may reflect their evolutionary position along the C3-C4 continuum. F. ramosissima, which possesses the most developed C3-C4 intermediate characteristics, maintained relatively stable mesophyll conductance under elevated CO2 while substantially reducing stomatal conductance. This response pattern suggests that the CO2-concentrating mechanism in this species provides sufficient substrate for carboxylation even with reduced stomatal aperture. The shift in limitation partitioning from stomatal to mesophyll dominance under elevated CO2 has important implications for understanding photosynthetic acclimation. Under ambient conditions, stomatal limitations accounted for the largest fraction of total diffusional resistance in all species, consistent with observations in typical C3 plants. However, the relative increase in mesophyll limitation under elevated CO2 suggests that anatomical or biochemical factors affecting CO2 transfer within the leaf become increasingly important when stomatal conductance is reduced. The maintenance of similar Ci/Ca ratios between ambient and elevated CO2 treatments, particularly in F. ramosissima, indicates coordination between stomatal responses and photosynthetic capacity that optimizes water use efficiency while maintaining adequate CO2 supply. This coordination may represent an important adaptive mechanism that allows intermediate species to benefit from elevated CO2 without excessive stomatal closure that could compromise carbon gain.

Discussion

The results of this research demonstrate that C3-C4 intermediate species exhibit distinct patterns of stomatal and mesophyll acclimation to elevated CO2 that differ from typical C3 plant responses. The reduction in stomatal conductance observed across all species is consistent with the general pattern reported for C3 plants, but the magnitude of reduction and its relationship to mesophyll conductance changes varied substantially among species. This variation likely reflects differences in the degree of C3-C4 intermediacy and the associated anatomical and biochemical modifications present in each species. The relatively stable mesophyll conductance in F. ramosissima under elevated CO2 is particularly noteworthy, as it suggests that the structural modifications associated with C3-C4 intermediacy may buffer mesophyll conductance against the reductions typically observed in C3 plants. The enlarged bundle sheath cells and altered mesophyll cell arrangement in intermediate species may facilitate CO2 diffusion even under conditions of reduced CO2 supply from stomata. This anatomical advantage could contribute to the maintenance of high carbon assimilation rates under water-limited conditions when stomatal conductance is reduced. The increase in electron transport rates under elevated CO2 without proportional increases in maximum electron transport capacity suggests that photosynthetic capacity under ambient conditions is not fully realized due to CO2 supply limitations. Under elevated CO2, the relaxation of these limitations allows increased flux through the electron transport chain, although the relatively unchanged Jmax values indicate that the potential for further enhancement may be limited by the regeneration capacity for RuBP. The findings from this research have implications for understanding how C3-C4 intermediate species may respond to continued increases in atmospheric CO2 concentrations. The ability of some intermediate species to maintain mesophyll conductance while reducing stomatal conductance could provide advantages in water use efficiency without sacrificing carbon assimilation capacity. This response pattern may partially explain the ecological success of C3-C4 intermediates in semi-arid environments where water availability constrains productivity.

Evolutionary Implications

The differential responses observed among the C3-C4 intermediate species investigated provide insights into the selective pressures that may have driven the evolution of C4 photosynthesis. The partial decoupling of stomatal and mesophyll conductance responses in species with more developed intermediate characteristics suggests that the CO2-concentrating mechanism provides functional benefits under conditions of reduced stomatal aperture. This advantage would be particularly important in water-limited environments where maintaining stomatal opening for carbon acquisition incurs significant water costs. The ability of C3-C4 intermediates to maintain favorable Ci/Ca ratios under both ambient and elevated CO2 conditions indicates robust homeostatic regulation of internal CO2 concentration. This regulatory capacity may represent an important pre-adaptation for the evolution of full C4 photosynthesis, which requires even more precise coordination between CO2 supply and demand to prevent overcycling of the C4 metabolic pathway.

Conclusion

This research provides comprehensive evidence that C3-C4 intermediate species possess distinct physiological mechanisms for responding to elevated atmospheric CO2

concentrations. The three species investigated demonstrated species-specific patterns of stomatal and mesophyll conductance adjustment that reflect their varying positions along the C3-C4 evolutionary continuum. These findings contribute to our understanding of how photosynthetic diversity influences plant responses to environmental change and have implications for predicting ecosystem function under future atmospheric conditions. The key findings from this research include: (1) This finding has implications for understanding the selective advantages that drove the evolution of C4 photosynthesis. The shift in limitation partitioning observed under elevated CO2 indicates that mesophyll factors become increasingly important determinants of photosynthetic capacity when stomatal conductance is reduced. This shift has implications for crop improvement strategies aimed at enhancing productivity under elevated CO2, as efforts focused solely on stomatal traits may be insufficient to realize potential gains in carbon assimilation. Instead, attention to mesophyll anatomy and the factors controlling CO2 transfer within leaves may be necessary to optimize photosynthetic performance. Future research should investigate the molecular and anatomical basis for the species-specific mesophyll conductance responses observed in this investigation. Understanding the genetic determinants of these responses could facilitate the introduction of beneficial traits into crop species through breeding or biotechnological approaches. Additionally, longer-term experiments examining acclimation responses over multiple generations would provide insights into the potential for evolutionary adaptation to elevated CO2 in intermediate species.

In conclusion, C3-C4 intermediate species exhibit complex and species-specific responses to elevated CO2 that involve coordinated adjustments in stomatal and mesophyll conductance. These responses suggest that the partial CO2concentrating mechanism present in intermediate species provides functional benefits under elevated atmospheric CO2 conditions, potentially conferring adaptive advantages in water-limited environments where stomatal closure is necessary to maintain water balance.

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