Low relative air humidity and increased stomatal density independently hamper growth in young Arabidopsis.

Stomatal pores in plant leaves mediate CO2 uptake for photosynthesis and water loss via transpiration. Altered stomatal density can affect plant photosynthetic capacity, water use efficiency, and growth, potentially providing either benefits or drawbacks depending on the environment. Here we explore, at different air humidity regimes, gas exchange, stomatal anatomy, and growth of Arabidopsis lines designed to combine increased stomatal density (epf1, epf2) with high stomatal sensitivity (ht1-2, cyp707a1/a3). We show that the stomatal density and sensitivity traits combine as expected: higher stomatal density increases stomatal conductance, whereas the effect is smaller in the high stomatal sensitivity mutant backgrounds than in the epf1epf2 double mutant. Growth under low air humidity increases plant stomatal ratio with relatively more stomata allocated to the adaxial epidermis. Low relative air humidity and high stomatal density both independently impair plant growth. Higher evaporative demand did not punish increased stomatal density, nor did inherently low stomatal conductance provide any protection against low relative humidity. We propose that the detrimental effects of high stomatal density on plant growth at a young age are related to the cost of producing stomata; future experiments need to test if high stomatal densities might pay off in later life stages.

Stomatal CO2 responses at sub- and above-ambient CO2 levels employ different pathways in Arabidopsis.

Stomatal pores that control plant CO2 uptake and water loss affect global carbon and water cycles. In the era of increasing atmospheric CO2 levels and vapor pressure deficit (VPD), it is essential to understand how these stimuli affect stomatal behavior. Whether stomatal responses to sub-ambient and above-ambient CO2 levels are governed by the same regulators and depend on VPD remains unknown. We studied stomatal conductance responses in Arabidopsis (Arabidopsis thaliana) stomatal signaling mutants under conditions where CO2 levels were either increased from sub-ambient to ambient (400 ppm) or from ambient to above-ambient levels under normal or elevated VPD. We found that guard cell signaling components involved in CO2-induced stomatal closure have different roles in the sub-ambient and above-ambient CO2 levels. The CO2-specific regulators prominently affected sub-ambient CO2 responses, whereas the lack of guard cell slow-type anion channel SLOW ANION CHANNEL-ASSOCIATED 1 (SLAC1) more strongly affected the speed of above-ambient CO2-induced stomatal closure. Elevated VPD caused lower stomatal conductance in all studied genotypes and CO2 transitions, as well as faster CO2-responsiveness in some studied genotypes and CO2 transitions. Our results highlight the importance of experimental setups in interpreting stomatal CO2-responsiveness, as stomatal movements under different CO2 concentration ranges are controlled by distinct mechanisms. Elevated CO2 and VPD responses may also interact. Hence, multi-factor treatments are needed to understand how plants integrate different environmental signals and translate them into stomatal responses.

Mitogen-activated protein kinases MPK4 and MPK12 are key components mediating CO2 -induced stomatal movements.

Respiration in leaves and the continued elevation in the atmospheric CO2 concentration cause CO2 -mediated reduction in stomatal pore apertures. Several mutants have been isolated for which stomatal responses to both abscisic acid (ABA) and CO2 are simultaneously defective. However, there are only few mutations that impair the stomatal response to elevated CO2 , but not to ABA. Such mutants are invaluable in unraveling the molecular mechanisms of early CO2 signal transduction in guard cells. Recently, mutations in the mitogen-activated protein (MAP) kinase, MPK12, have been shown to partially impair CO2 -induced stomatal closure. Here, we show that mpk12 plants, in which MPK4 is stably silenced specifically in guard cells (mpk12 mpk4GC homozygous double-mutants), completely lack CO2 -induced stomatal responses and have impaired activation of guard cell S-type anion channels in response to elevated CO2 /bicarbonate. However, ABA-induced stomatal closure, S-type anion channel activation and ABA-induced marker gene expression remain intact in the mpk12 mpk4GC double-mutants. These findings suggest that MPK12 and MPK4 act very early in CO2 signaling, upstream of, or parallel to the convergence of CO2 and ABA signal transduction. The activities of MPK4 and MPK12 protein kinases were not directly modulated by CO2 /bicarbonate in vitro, suggesting that they are not direct CO2 /bicarbonate sensors. Further data indicate that MPK4 and MPK12 have distinguishable roles in Arabidopsis and that the previously suggested role of RHC1 in stomatal CO2 signaling is minor, whereas MPK4 and MPK12 act as key components of early stomatal CO2 signal transduction.