Amphistomatic leaf surfaces independently regulate gas exchange in response to variations in evaporative demand.

The occurrence of amphistomatic leaves (stomata on both surfaces) versus hypostomatic leaves (stomata limited to the lower or abaxial surface) has strong associations with environment. Amphistomy provides the advantage of higher conductance of CO2 for photosynthesis, however, unless the stomata on both leaf surfaces can be independently controlled in response to environmental cues, amphistomy may lead to inefficient gas exchange. While previous studies have found evidence that stomata can operate independently across and between surfaces of dorsiventral leaves, we investigate whether an independent stomatal response can be induced for isobilateral leaves by largely natural conditions. Here, we exposed surfaces of isobilateral, amphistomatic Eucalyptus globulus Labill. leaves to natural diurnal variation in differential evaporative demand, using leaf orientation to drive differences in irradiance and heat load on leaf surfaces. We identified preferential closure of stomata on the surface exposed to higher irradiation (and therefore evaporative demand) during the afternoon under natural conditions and similarly induced differential stomatal closure under experimental conditions in the laboratory. The differential response confirms that sufficient hydraulic isolation exists for independent stomatal response to occur between surfaces of amphistomatic, isobilateral leaves, and importantly, we show that natural conditions can induce surface-specific stomatal closure.