Major diffusion leaks of clamp-on leaf cuvettes still unaccounted: how erroneous are the estimates of Farquhar et al. model parameters?

Estimates of leaf gas-exchange characteristics using standard clamp-on leaf chambers are prone to errors because of diffusion leaks.While some consideration has been given to CO2 diffusion leaks, potential water vapour diffusion leaks through chamber gaskets have been neglected. We estimated diffusion leaks of two clamp-on Li-Cor LI-6400 (Li-Cor, Inc., Lincoln, NE, USA) leaf chambers with polymer foam gaskets and enclosing either 2 or 6 cm2 leaf area, and conducted a sensitivity analysis of the diffusion leak effects on Farquhar et al. photosynthesis model parameters – the maximum carboxylase activity of ribulose 1·5-bisphosphate carboxylase/oxygenase (Rubisco) (Vcmax), capacity for photosynthetic electron transport (Jmax) and nonphotorespiratory respiration rate in light (Rd). In addition, net assimilation rate (An) versus intercellular CO2 (Ci) responses were measured in leaves of Mediterranean evergreen species Quercus ilex L. enclosing the whole leaf chamber in a polyvinyl fluoride bag flushed with the exhaust air of leaf chamber, thereby effectively reducing the CO2 and water vapour gradients between ambient air and leaf chamber. For the empty chambers, average diffusion leak for CO2, KCO2 , (molar flow rate corresponding to unit CO2 mole fraction difference) was ca. 0.40 mmol s-1. KCO2 increased ca. 50% if a dead leaf was clamped between the leaf chamber. Average diffusion leak for H2O was ca. 5- to 10-fold larger than the diffusion leak for CO2. Sensitivity analyses demonstrated that the consequence of a CO2 diffusion leak was apparent enhancement of An at high CO2 mole fraction and reduction at lower CO2 mole fraction, and overall compression of Ci range. As the result of these modifications, Farquhar et al. model parameters were overestimated. The degree of overestimation increased in the order of Vcmax < Jmax < Rd, and was larger for smaller chambers and for leaves with lower photosynthetic capacity, leading to overestimation of all three parameters by 70–290% for 2 cm2, and by 10–60% for 6 cm2 chamber. Significant diffusion corrections (5–36%) were even required for leaves with high photosynthetic capacity measured in largest chamber. Water vapour diffusion leaks further enhanced the overestimation of model parameters. For small chambers and low photosynthetic capacities, apparent Ci was simulated to decrease with increasing An because of simultaneous CO2 and H2O diffusion leaks. Measurements in low photosynthetic capacity Quercus ilex leaves enclosed in 2 cm2 leaf chamber exhibited negative apparent Ci values at highest An. For the same leaves measured with the entire leaf chamber enclosed in the polyvinyl fluoride bag, Ci and An increased monotonically. While the measurements without the bag could be corrected for diffusion leaks, the required correction in An and transpiration rates was 100–500%, and there was large uncertainty in Farquhar et al. model parameters derived from ‘corrected’ An/Ci response curves because of uncertainties in true diffusion leaks. These data demonstrate that both CO2 and water vapour diffusion leaks need consideration in measurements with clamp-on leaf cuvettes. As plants in natural environments are often characterized by low photosynthetic capacities, cuvette designs need to be improved for reliable measurements in such species.