Stomatal density and gas exchange efficiency in C3 and C4 grasses under elevated CO2 conditions

Introduction

Keeping pace with rising atmospheric CO 2 is one of the defining challenges for plant biology in the 21st century. Since pre-industrial times, CO 2 concentrations have climbed from roughly 280 ppm to over 420 ppm, and projections suggest levels could reach 700-800 ppm by 2100 under high-emission scenarios [1] . Plants respond to elevated CO 2 through a range of mechanisms, but one of the most visible is a reduction in stomatal density-the number of stomatal pores per unit leaf area [2] . Fewer stomata mean reduced water loss through transpiration, but also altered CO 2 uptake kinetics. The magnitude of this response differs markedly between C3 and C4 photosynthetic pathways [3] . C3 grasses, which fix carbon directly through RuBisCO, tend to show larger responses to elevated CO 2 because their photosynthesis is often CO 2 -limited under current atmospheric concentrations. C4 grasses, with their carbon-concentrating mechanism around RuBisCO, are already operating near CO 2 saturation and may respond less dramatically [4] . Yet direct side-by-side comparisons of stomatal and gas exchange responses in C3 versus C4 grasses grown under identical controlled conditions remain surprisingly few [5] . This research aimed to compare stomatal density, gas exchange parameters, and water use efficiency between three C3 and three C4 grass species across three CO 2 levels.

Materials and Methods

Materials

This research was conducted at the Department of Plant Physiology, Hokuriku College of Biological Research, Kanazawa, Ishikawa, Japan, from June 2023 to August 2024. Institutional approval was obtained (HCBR-PC/2023-019). Seeds of six grass species were germinated in plug trays and transferred to 3-liter pots containing a standardized potting mix. Plants were grown in three walk-in controlled environment chambers (Koito Industries, Japan) set to 400, 600, or 800 ppm CO 2 , with temperature at 25/18 °C (day/night), 14-hour photoperiod, and 60% relative humidity.

Each species-CO 2 combination had 10 replicate pots. Plants were watered to field capacity every two days.

Methods

Stomatal density was measured on the abaxial surface of the third fully expanded leaf at 8 weeks post-transplant. Clear nail varnish impressions were examined under a compound microscope at 400× magnification, and stomata were counted in five fields of view per leaf on three leaves per plant [2] . Gas exchange measurements used a LI-6400XT portable photosynthesis system (LI-COR, Lincoln, NE, USA) at growth CO 2 concentration, photosynthetically active radiation of 1500 μmol m⁻² s⁻¹, and leaf temperature of 25 °C [6] . Parameters recorded included net photosynthetic rate (Anet), stomatal conductance (gs), and intercellular CO 2 concentration (Ci). Intrinsic water use efficiency was calculated as Anet/gs. Data were analyzed by two-way ANOVA (species × CO 2 ) with Tukey's HSD posthoc test using R version 4.3 [7] . C3 grasses showed a 28.3% decline in stomatal density from 400 to 800 ppm (p<0.001), while C4 grasses declined by only 12.7% (p = 0.014). Net photosynthesis in C3 species increased by 31.4% across the CO 2 range (p<0.001) but didn't change significantly in C4 species (p = 0.412).

Results

Discussion

The stronger stomatal density response in C3 grasses compared to C4 grasses is consistent with the expectation that C3 species, being more CO 2 -limited under ambient conditions, have more to gain from reduced stomatal aperture under elevated CO 2 [3,4] . The 28.3% reduction in C3 stomatal density agrees with meta-analyses reporting 20-35% decreases under doubled CO 2 [2] . C4 grasses already maintain low Ci through their carbon-concentrating mechanism, so the additional CO 2 provides little photosynthetic benefit and correspondingly less stimulus for stomatal reduction [5] . The substantial improvement in water use efficiency across both groups has practical implications for grassland management under climate changeparticularly in water-limited environments where higher WUE could buffer against drought stress [1] . But the differential magnitude of response suggests that C3 grasses may gain a competitive edge under elevated CO 2 in mixedspecies grasslands, potentially shifting community composition over time [8] .

Conclusion

This research demonstrated that C3 grasses reduce stomatal density more sharply than C4 grasses under elevated CO 2 , with correspondingly larger gains in net photosynthesis and water use efficiency. C4 grasses showed modest stomatal responses with minimal photosynthetic stimulation, consistent with their carbon-concentrating mechanism buffering against CO 2 changes. These differential responses have implications for predicting shifts in grassland community composition under future atmospheric conditions and may inform breeding strategies for climateadapted forage grasses.

Acknowledgement

Acknowledgements Funding SourcesInternal research funds from the Hokuriku College of Biological Research supported this work. Institutional SupportThe authors thank the Hokuriku College of Biological Research for controlled environment chamber access and gas exchange equipment. Contributions Not Qualifying for AuthorshipGreenhouse staff who maintained chamber conditions are gratefully acknowledged.

References

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