Elevated CO₂ promotes satellite stomata production in young cotyledons of Arabidopsis thaliana

Stomata are tiny pores on plant leaves and stems surrounded by a pair of differentiated epidermal cells known as guard cells. Plants undergo guard cell differentiation in response to environmental cues, including atmospheric CO₂ . To quantitatively evaluate stomatal development in response to elevated CO₂ , imaging analysis of stomata was conducted using young cotyledons of Arabidopsis thaliana grown under ambient (380 ppm) and elevated (1,000 ppm) CO₂ conditions. Our analysis revealed that treatment with 1,000 ppm CO₂ did not affect stomatal numbers on abaxial sides of cotyledons but increased cotyledon area, resulting in decreased stomatal density, 7 days after germination. Interestingly, this treatment also perturbed the uniform distribution of stomata via excess satellite stomata and stomatal precursor cells. We used overexpression lines of the DNA replication licensing factor gene CDC6, a reported positive regulator of satellite stomata production. CDC6 overexpression decreased the speed of cotyledon expansion, even under treatment with 1,000 ppm CO₂ , possibly by suppressing pavement cell maturation. In contrast, treatment with 1,000 ppm CO₂ induced stomatal distribution changes in the overexpressor. These results suggest that treatment with 1,000 ppm CO₂ enhances both cotyledon expansion and satellite stomata production via independent pathways, at least in young cotyledons of A. thaliana.