Effects of a 4-year exposure to pre-industrial and elevated CO₂ gradient on stomatal characters of C₃ and C₄ species

Abstract

Many studies report that past increase in atmospheric [CO₂] are associated with reduced stomatal density (SD). However, many of these studies are based on paleofossils or herbarium specimens where other environmental factors may affect stomatal distribution. To understand the effect of CO₂ on stomatal distribution, we examined 9 grassland species after 4 years of growth in the field under an experimental gradient of CO₂ ranging from pre-industrial to elevated concentrations (200 to 550 µmol CO₂ mol^-1). In the field, casts were made on leaves of C₃ and C₄ annual and perennial grasses and forbs. Leaf impressions were made from the casts and SD, stomatal index (SI), and stomatal aperture length (AP) were measured. Individually, the C₄ grasses showed no strong negative linear relationship between SD and [CO₂], SI and [CO₂], or AP and [CO₂]. In contrast, C₃ annuals showed strong positive and negative linear relationships between SD and [CO₂] or SI and [CO₂]. One C₃ perennial, Solanum dimidiatum, had a strong positive linear relationship between SD and [CO₂] that was associated with a negative relationship between AP and [CO₂]. In all species, AP either decreased or was unchanged by [CO₂]. These stomatal parameters show no effect of CO₂ on C₄ grasses but different responses on C₃ species, suggesting limited use of preserved material as [CO₂] proxy. Also, C₃ plants respond to CO₂ by adjusting stomatal cell initiation, epidermal cell expansion, or both.

Objective

Determine the long-term effects of CO₂ on the stomatal frequency of different species grown along a gradient of pre-industrial to projected future CO₂.

Experimental design

• Full-sun mature leaves from 9 species were sampled for stomatal frequencies.
  • 3 C₃ Annuals: Bromus japonicus (BR), Croton monanthogynous (CM), Euphorbia bicolor (Eubi)
  • 3 C₃ Perennials: Convovulus equitans (CoEq), Solanum dimidiatum (SD), Solidago canadensis (SC)
  • 3 C₄ Perennials: Bothriochloa ischaemum (KR), Paspalum pubescens (Ppa), Sorghum halepense (Sha)
  • Species were sampled at different periods according to their phenology
  • Individual leaf area estimated from leaf length and width.
• Stomatal impressions:
  • Casts of the leaves were made in the field using dental impression material (Kerr Extrude-Medium).
  • Leaf impressions were made from clear nail polish peels of the cast.
  • Number and length of stomata, and number of epidermal cells were recorded under a light microscope hooked up to a computer imaging system.
• Stomatal frequency calculated by:
  • Stomatal density (SD) = # stomata mm^-2
  • Epidermal cell counts (ECD) = # epidermal cells mm^-2
  • Stomatal index (SI) = [SD / (SD+ECD)] *100

Hypothesis

Stomatal frequency decreases with increasing atmospheric [CO₂]

‘An old hypothesis revisited’

• Körner et al. (1986) observed changes in stomatal density with changing pCO₂ in altitudinal gradients.
• Woodward’s (1987) seminal paper reported a decrease in stomatal density with increasing CO₂ from pre-industrial to current levels.

But

• Evidence from elevated CO₂ experiments does not always support it.
• Evidence from natural CO₂ vents gives variable support (Bettarini et al. 1998).
• Our earlier research with multi-generations of Arabidopsis at different CO₂ did not support it.
   
• Yet, the hypothesis is now used to reconstruct past CO₂ environments from paleofossils (McElwain et al. 1999)
• As stomatal characters are a main determinant of water use and are correlated with plant tolerance to pollutants, such understanding will help predict water use in future environments and guide breeding efforts for increased pollutant tolerance.

Field chambers on a C₃/C₄ grassland in Temple, TX

• Two tunnel-shaped field chambers
  • A 560 to 350 µmol CO₂ mol ^-1 tunnel
  • A 365 to 200 µmol CO₂ mol ^-1 tunnel
• Gradients of CO₂ are driven by daytime photosynthesis and nighttime respiration (Johnson et al. 2000)

Tunnel sections controlled at different CO₂ levels

• Air flow is adjusted to maintain preset [CO₂]
• Watering is adjusted to simulate rainfall outside
• Sealed slits are used to sample with mimimum disturbance

Leaf impressions at 400x

C₃
Annuals

C₃
Perennials

C₄
Perennials

Conclusions

• From pre-industrial to elevated CO₂, only 2 of 9 species had a significant decrease in SD and 1 of 9 species had a significant decrease in SI, in contrast to our hypothesis.
• Two of the 9 species, B. japonicus and S. dimidiatum, had a significant increase in SD. For S. dimidiatum, the increase in SD was compensated by a decrease in the size of the stomata.
• Stomatal size is decreased with increased CO₂ suggesting that the reduction in leaf conductance is by a combination of change in frequency, anatomy and function.