Advancing the helox compensation method to estimate intercellular humidity in leaves of diverse species

The relative humidity in the intercellular airspaces of leaves (h) has long been thought to be saturating, but several recent studies have questioned this assumption. We present and develop a method to estimate h, describe practical considerations for its application, and demonstrate it on eight species (Gossypium hirsutum, Helianthus annuus, Heteromeles arbutifolia, Laurus nobilis, Liriodendron tulipifera, Magnolia grandiflora, Oryza sativa, and Pyrrosia hastata). N₂/O₂ (nitox) is replaced with He/O₂ (helox), which speeds stomatal diffusion, but ambient humidity is simultaneously increased to keep transpiration rate unchanged. This allows the expressions for transpiration in nitox and helox to be set equal to one another, leading to a solution for h. We were able to rapidly switch to helox and simultaneously change humidity such that transpiration rate remained unchanged to within ±2.5% (< 0.54% in most cases). We found h ranging between 71 and 94% of saturation. Whereas other methods to estimate h either require special analytical tools (isotopic gas analysis, spectroscopically resolved fluorescence measurements with proprietary fluorophores) or special conditions (amphistomatous broad leaves with zero CO₂ exchange at one leaf surface), our method requires only water vapor concentration measurements and can be performed on any leaf under nearly any conditions.