Design and fabrication of an improved dynamic flow cuvette for 13CO2 labeling in Arabidopsis plants

Abstract

Background: Stable isotope labeling is a non-invasive, sensitive means of monitoring metabolic flux in plants. The most physiologically meaningful information is obtained from experiments that take advantage of the natural photosynthetic carbon assimilation pathway to introduce a traceable marker with minimal effects on the physiology of the organism. The fundamental substrate in isotopic labeling experiments is 13CO2, which can reveal the earliest events in carbon assimilation and realistically portray downstream metabolism when administered under conditions suitable for making kinetic inferences. Efforts to improve the accuracy and resolution of whole plant labeling techniques have focused on improvements in environmental control, air flow characteristics, and harvesting methods. Results: Here we present a dynamic flow cuvette designed for single Arabidopsis thaliana labeling experiments. We have also verified its suitability for labeling Nicotiana benthamiana and essential oils in Pelargonium graveolens. Complete plans for fabrication of this device are included. The design includes three important innovations. First, uniform, circular air flow over the rosette surface is accomplished by a fan and deflector that creates a mini-cyclone effect within the chamber interior. Second, a network of circulating canals connected to a water bath provides temperature control to within ± 0.1 ºC under variable irradiance, humidity, and air flow conditions. When photosynthetically active radiation (PAR) was varied over a range of 1000 μEinsteins m−2 s−1 with no adjustment to the external temperature control system, the abaxial leaf temperature changed by < 3 ºC/1000 PAR. Third, the device is fully compatible with liquid nitrogen quenching of metabolic activity without perturbation of the light environment. For short labeling experiments (< 10 s), the most critical variable is the half-life (t1/2) of the atmosphere within the chamber, which determines the maximum resolution of the labeling system. Using an infrared gas analyzer, we monitored the atmospheric half-life during the transition from 12CO2 to 13CO2 air at different flow rates and determined that 3.5 L min−1 is the optimal flow rate to initiate labeling (t1/2 ~ 5 s). Under these conditions, we observed linear incorporation of 13C into triose phosphate with labeling times as short as 5 s. Conclusions: Advances in our ability to conduct short term labeling experiments are critical to understanding of the rates and control of the earliest steps in plant metabolism. Precise kinetic measurements in whole plants using 13CO2 inform metabolic models and reveal control points that can be exploited in agricultural or biotechnological contexts. The dynamic labeling cuvette presented here is suitable for studying early events in carbon assimilation and provides high resolution kinetic data for studies of metabolism in intact plants under physiologically realistic scenarios.