Miniaturised carbon dioxide sensor designed for measurements within plant leaves

To permit CO2 measurements behind stomatal pores of plant leaves a potentiometric CO2-biosensor was built with a tip diameter of 2 mum. The miniaturised sensor assembly consists of a H+ carrier-based pH-microelectrode concentrically arranged within a sheathing micropipette. The tip of the latter is filled with carbonate buffer, the pH of which quickly responds to CO2 concentration changes due to incorporated carbonic anhydrase. The CO2-microsensor shows a linear response to CO2 in the physiologically relevant concentration range of 50-800 ppm (5.1-81.1 Pa). The 90% response time varied from sensor to sensor between 18 and 63 s (mean S.E., 42 +/- 14 s; n = 6). Sensor calibration and leaf experiments were performed in an open-flow tube-like minicuvette, allowing tangential air flow along the leaf surface with controlled gas mixtures and flow rates of choice. At 800 ppm external CO2 concentration, CO2 within the leaf was close to the external CO2 concentration when stomatal pores were wide open. However, the concentration dropped to 350 ppm during stomatal closure due to CO2 consumption by photosynthesis demonstrating distinct CO,sensing of internal leaf CO2. Following 'light-off' internal CO2 rapidly rose close to 700 ppm, a response which was completely reversed by 'light-on'. We conclude that this sensor is a suitable tool for CO2 monitoring in places too small to be accessible to conventional tools. (C) 2001 Elsevier Science B.V. All rights reserved.