Ethylene biosynthesis in guard cells, not mesophyll, predominantly drives stomatal conductance responses to CO 2

Summary Research and rationale : This study investigates whether tissue-specific ethylene biosynthesis regulates stomatal conductance (g s ) responses to changing [CO₂] in Arabidopsis thaliana . While guard cells sense [CO₂], mesophyll-derived signals are also implicated in stomatal control. We aimed to determine if ethylene production in guardcells or mesophyll is the primary driver of CO₂-induced g s regulation. Methods: An acs octuple mutant with severely reduced ethylene production was complemented with tissue-specific ACS8/ACS11 transgenes driven by guard-cell, spongy-mesophyll, dual palisade/spongy-mesophyll, or whole-leaf promoters. Tissue-specific complementation in the different transgenic lines was confirmed and evaluated by qPCR, tissue-specific NEON expression, microscopic imaging, and ethylene production measurements. Gas-exchange measurements on intact plants recorded g s kinetics, CO 2 assimilation, and water-use efficiency, across CO₂ shifts. Key results : Guard-cell complementation nearly fully restored wild-type g s responses and reversed the mutant’s aberrant leaf phenotype. Spongy-mesophyll complementation failed to rescue either trait, while dual palisade- and spongy-mesophyll complementation yielded only partial recovery. Conclusion: Ethylene produced in guard cells is the dominant regulator of CO₂-induced stomatal conductance regulation, with mesophyll-derived ethylene contributing secondarily via long-distance signaling or by augmenting the overall ethylene pool. These findings underscore the importance of spatially regulated ethylene biosynthesis in balancing carbon assimilation and transpiration.