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

Andrew P. Scafaro, A. Clarissa A. Negrini, Brendan O'Leary, F. Azzahra Ahmad Rashid, Lucy Hayes, Yuzhen Fan, You Zhang, Vincent Chochois, Murray R. Badger, A. Harvey Millar, Owen K. Atkin

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13 Citations (Scopus)

Abstract

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 O2 consumption rates. The fluorophore technique was compared with O2-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.

Original languageEnglish
Article number16
JournalPlant Methods
Volume13
Issue number1
DOIs
Publication statusPublished - 21 Mar 2017

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    Scafaro, A. P., Negrini, A. C. A., O'Leary, B., Rashid, F. A. A., Hayes, L., Fan, Y., Zhang, Y., Chochois, V., Badger, M. R., Millar, A. H., & Atkin, O. K. (2017). The combination of gas-phase fluorophore technology and automation to enable high-throughput analysis of plant respiration. Plant Methods, 13(1), [16]. https://doi.org/10.1186/s13007-017-0169-3