Direct measurement of intercellular CO(2) concentration in a gas-exchange system resolves overestimation using the standard method

Intercellular CO(2) concentration of leaves (C(i)) is a critical parameter in photosynthesis. Nevertheless, uncertainties in calculating C(i) arise as stomata close. Here, by modifying the assimilation chamber of a commercial gas-exchange equipment to directly measure C(i), we demonstrate overestimation of calculated C(i) (i.e. C(i(c))) without stimulating stomatal closure. Gas exchange was measured on one side of the leaf while measured C(i) (C(i(m))) was acquired simultaneously on the other side of the leaf in hypostomatous passion fruit (Passiflora edulis Sims) and amphistomatous sunflower (Helianthus annuus L.) and common bean (Phaseolus vulgaris L.). The adaxial surface showed comparable C(i(c)) and C(i(m)) in sunflower, whereas in common bean, where the adaxial surface has a low stomatal density, C(i(c)) markedly differed from C(i(m)) when the stomata remained open. However, the latter discrepancy disappeared when measuring the leaf flipped upside down so that the gas exchange was measured (i.e. C(i) was calculated) on the abaxial side, which has a much higher stomatal density. The passion fruit showed the largest discrepancy on the astomatous side, indicating that the cuticle has a large impact on the calculation. Direct measurement of C(i) is recommended as a more accurate estimate than the calculation when stomatal gas transport is restricted. Occurrence of overestimation and prospects for direct measurement are discussed.