Carbon dioxide or photosynthetically active radiation? Evaluation of the significance of individual environmental factors that control leaf stomatal development

Background and Aims Stomata are pores in the plant epidermis that regulate gas exchange by controlling CO2 uptake and water loss through transpiration. Stomatal conductance, which determines the rate of this gas exchange, is influenced by both the dynamic aperture of individual stomata and stomatal density (StD). It is a matter of discussion whether a single dominant factor that controls StD exists or a combination of several independent factors results in the final StD. Methods We evaluated the role of photosynthetic photon flux density (PPFD) and internal CO2 concentration (Ci). Plants of Helianthus annuus were cultivated under a gradient of PPFD in the range of 50–510 μmol m−2 s−1; both cotyledons and true leaves were analysed. The independent effects of PPFD and Ci on stomatal traits were assessed using additive linear models controlling for their physiological covariation. Results The results demonstrate that stomatal development in sunflower leaves is predominantly regulated by PPFD, not Ci. While stomatal traits in cotyledons remained largely unaffected by either PPFD or Ci, true leaves showed a clear and consistent response to light intensity. Additive linear models confirmed that only PPFD had a significant and robust effect on StD, whereas Ci showed no consistent contribution. This supports the interpretation that Ci, although physiologically coupled with PPFD, does not directly control stomatal development. Furthermore, we observed anatomical asymmetry between leaf surfaces: while both adaxial and abaxial sides responded to PPFD, the abaxial surface showed higher sensitivity, with significant effects on both StD and stomatal length. Conclusions Our findings emphasize light as the key environmental signal guiding stomatal patterning. Additionally, the results revealed varying differences in stomatal development on adaxial and abaxial sides of the leaf in response to light.