Cuticle Affects Calculations of Internal CO2 in Leaves Closing Their Stomata

Analyzing the assimilation rate (A) relative to the CO(2) concentration inside leaves (C(i)) has been a useful approach for investigating plant responses to various environments. Nevertheless, there are uncertainties in calculating C(i) when stomata close, restricting the application. Here, A-C(i) curves were traced in sunflower (Helianthus annuus L.) leaves using a method for directly measuring C(i). The method was incorporated into an LI-6400 open gas exchange system, and stomata were closed by feeding 10 µM ABA through petioles. The conductance to CO(2) was derived from the directly measured C(i) and compared with the conductance from the water vapor flux (i.e. the standard calculation). When stomata were open, measured and calculated C(i) gave similar A-C(i) curves. When stomata were closed, the curves differed because measured C(i) departed from the calculated value. This difference caused the calculation to trace an artifactual limitation of photosynthesis. The direct measurement avoided this problem and followed the curve for leaves with open stomata. Largely because of the cuticle, the calculation overestimated CO(2) entry into the leaf because the cuticle transmitted more water vapor than CO(2), and the calculation relied on water vapor. Consequently, the standard calculation gave conductances larger than those from directly measured C(i). Although the cuticle conductance to water vapor remained constant as stomata closed, it increasingly contributed to the overestimation of C(i). The system provided here is not affected by these cuticle properties and thus is expected to open up the opportunity for A-C(i) analysis in plant physiology.