Guard cell photosynthesis is critical for stomatal turgor production, yet does not directly mediate CO2- and ABA-induced stomatal closing

Stomata mediate gas exchange between the inter-cellular spaces of leaves and the atmosphere. CO2 levels in leaves (Ci) are determined by respiration, photosynthesis, stomatal conductance and atmospheric [CO2]. [CO2] in leaves mediates stomatal movements. The role of guard cell photosynthesis in stomatal conductance responses is a matter of debate, and genetic approaches are needed. We have generated transgenic Arabidopsis plants that are chlorophyll-deficient in guard cells only, expressing a constitutively active chlorophyllase in a guard cell specific enhancer trap line. Our data show that more than 90% of guard cells were chlorophyll-deficient. Interestingly, approximately 45% of stomata had an unusual, previously not-described, morphology of thin-shaped chlorophyll-less stomata. Nevertheless, stomatal size, stomatal index, plant morphology, and whole-leaf photosynthetic parameters (PSII, qP, qN, F-V/F-M) were comparable with wild-type plants. Time-resolved intact leaf gas-exchange analyses showed a reduction in stomatal conductance and CO2-assimilation rates of the transgenic plants. Normalization of CO2 responses showed that stomata of transgenic plants respond to [CO2] shifts. Detailed stomatal aperture measurements of normal kidney-shaped stomata, which lack chlorophyll, showed stomatal closing responses to [CO2] elevation and abscisic acid (ABA), while thin-shaped stomata were continuously closed. Our present findings show that stomatal movement responses to [CO2] and ABA are functional in guard cells that lack chlorophyll. These data suggest that guard cell CO2 and ABA signal transduction are not directly modulated by guard cell photosynthesis/electron transport. Moreover, the finding that chlorophyll-less stomata cause a deflated' thin-shaped phenotype, suggests that photosynthesis in guard cells is critical for energization and guard cell turgor production. Significance Statement In this study, transgenic plants that lack chlorophyll specifically in guard cells were generated and analyzed. The results provide genetic evidence that CO2/ABA-induced stomatal closure is not directly mediated by guard cell photosynthesis/electron transport. Moreover, approximately 45% of the stomata in these lines are deflated showing a previously not-described thin-shaped' stomatal morphology, which suggests a key function of guard cell photosynthesis for energization and turgor production of stomatal guard cells.