Ethylene biosynthesis in guard cells, not mesophyll, predominantly drives stomatal conductance responses to CO2

O_LIResearch and rationale: This study investigates whether tissue-specific ethylene biosynthesis regulates stomatal conductance (gs) 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 gs regulation. C_LIO_LIMethods: 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 gs kinetics, CO2 assimilation, and water-use efficiency, across CO2 shifts. C_LIO_LIKey results: Guard-cell complementation nearly fully restored wild-type gs responses and reversed the mutants aberrant leaf phenotype. Spongy-mesophyll complementation failed to rescue either trait, while dual palisade- and spongy-mesophyll complementation yielded only partial recovery. C_LIO_LIConclusion: 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. C_LI