The combination of gas-phase fluorophore technology and automation to enable high-throughput analysis of plant respiration

BACKGROUND: Mitochondrial respiration in the dark (R (dark)) is a critical plant physiological process, and hence a reliable, efficient and high-throughput method of measuring variation in rates of R (dark) is essential for agronomic and ecological studies. However, currently methods used to measure R (dark) in plant tissues are typically low throughput. We assessed a high-throughput automated fluorophore system of detecting multiple O(2) consumption rates. The fluorophore technique was compared with O(2)-electrodes, infrared gas analysers (IRGA), and membrane inlet mass spectrometry, to determine accuracy and speed of detecting respiratory fluxes. RESULTS: The high-throughput fluorophore system provided stable measurements of R (dark) in detached leaf and root tissues over many hours. High-throughput potential was evident in that the fluorophore system was 10 to 26-fold faster per sample measurement than other conventional methods. The versatility of the technique was evident in its enabling: (1) rapid screening of R (dark) in 138 genotypes of wheat; and, (2) quantification of rarely-assessed whole-plant R (dark) through dissection and simultaneous measurements of above- and below-ground organs. DISCUSSION: Variation in absolute R (dark) was observed between techniques, likely due to variation in sample conditions (i.e. liquid vs. gas-phase, open vs. closed systems), indicating that comparisons between studies using different measuring apparatus may not be feasible. However, the high-throughput protocol we present provided similar values of R (dark) to the most commonly used IRGA instrument currently employed by plant scientists. Together with the greater than tenfold increase in sample processing speed, we conclude that the high-throughput protocol enables reliable, stable and reproducible measurements of R (dark) on multiple samples simultaneously, irrespective of plant or tissue type. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13007-017-0169-3) contains supplementary material, which is available to authorized users.