Mesophyll airspace unsaturation drives C₄ plant success under vapor pressure deficit stress

A fundamental assumption in plant science posits that leaf air spaces remain vapor saturated, leading to the predominant view that stomata alone control leaf water loss. This concept has been pivotal in photosynthesis and water-use efficiency research. However, recent evidence has refuted this longstanding assumption by providing evidence of unsaturation in the leaf air space of C₃ plants under relatively mild vapor pressure deficit (VPD) stress. This phenomenon represents a nonstomatal mechanism restricting water loss from the mesophyll. The potential ubiquity and physiological implications of this phenomenon, its driving mechanisms in different plant species and habitats, and its interaction with other ecological adaptations have. In this context, C₄ plants spark particular interest for their importance as crops, bundle sheath cells' unique anatomical characteristics and specialized functions, and notably higher water-use efficiency relative to C₃ plants. Here, we confirm reduced relative humidities in the substomatal cavity of the C₄ plants maize, sorghum, and proso millet down to 80% under mild VPD stress. We demonstrate the critical role of nonstomatal control in these plants, indicating that the role of the CO₂ concentration mechanism in CO₂ management at a high VPD may have been overestimated. Our findings offer a mechanistic reconciliation between discrepancies in CO₂ and VPD responses reported in C₄ species. They also reveal that nonstomatal control is integral to maintaining an advantageous microclimate of relatively higher CO₂ concentrations in the mesophyll air space of C₄ plants for carbon fixation, proving vital when these plants face VPD stress.