Measurement of Gross Photosynthesis, Respiration in the Light, and Mesophyll Conductance Using H218O Labeling

A fundamental challenge in plant physiology is independently determining the rates of gross O₂ production by photosynthesis and O₂ consumption by respiration, photorespiration, and other processes. Previous studies on isolated chloroplasts or leaves have separately constrained net and gross O₂ production (NOP and GOP, respectively) by labeling ambient O₂ with ¹⁸O while leaf water was unlabeled. Here, we describe a method to accurately measure GOP and NOP of whole detached leaves in a cuvette as a routine gas-exchange measurement. The petiole is immersed in water enriched to a δ¹⁸O of ∼9,000‰, and leaf water is labeled through the transpiration stream. Photosynthesis transfers ¹⁸O from H₂O to O₂ GOP is calculated from the increase in δ¹⁸O of O₂ as air passes through the cuvette. NOP is determined from the increase in O₂/N₂ Both terms are measured by isotope ratio mass spectrometry. CO₂ assimilation and other standard gas-exchange parameters also were measured. Reproducible measurements are made on a single leaf for more than 15 h. We used this method to measure the light response curve of NOP and GOP in French bean (Phaseolus vulgaris) at 21% and 2% O₂ We then used these data to examine the O₂/CO₂ ratio of net photosynthesis, the light response curve of mesophyll conductance, and the apparent inhibition of respiration in the light (Kok effect) at both oxygen levels. The results are discussed in the context of evaluating the technique as a tool to study and understand leaf physiological traits.