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

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

doi:10.1186/s13007-017-0169-3

Abstract

BackgroundMitochondrial respiration in the dark (Rdark) is a critical plant physiological process, and hence a reliable, efficient and high-throughput method of measuring variation in rates of Rdark is essential for agronomic and ecological studies. However, currently methods used to measure Rdark 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.ResultsThe high-throughput fluorophore system provided stable measurements of Rdark 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 Rdark in 138 genotypes of wheat; and, (2) quantification of rarely-assessed whole-plant Rdark through dissection and simultaneous measurements of above- and below-ground organs.DiscussionVariation in absolute Rdark 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 Rdark 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 Rdark on multiple samples simultaneously, irrespective of plant or tissue type.

Highlights

Mitochondrial respiration in the dark (Rdark) is a critical plant physiological process, and a reliable, efficient and high-throughput method of measuring variation in rates of rates in the dark (Rdark) is essential for agronomic and ecological studies
To illustrate the potential of the high-throughput fluorophore technology to accelerate our understanding of plant Rdark, we report on: (1) a screen of Rdark in 138 genotypes of wheat that was conducted over a few days; and, (2) rapid assessments of respiration in leaf, stem and root tissues that enable whole-plant respiratory fluxes to be estimated by simultaneous analysis of individually dissected plants
When cut leaf material was placed inside the sample tubes, the fluorophore system was able to measure a consistent decline in O2 over a greater than 7-h period following an initial 1 h period of stabilization (Fig. 1a)

Summary

Introduction

Mitochondrial respiration in the dark (Rdark) is a critical plant physiological process, and a reliable, efficient and high-throughput method of measuring variation in rates of Rdark is essential for agronomic and ecological studies. There is a growing need to describe and predict variability in rates of plant R, which in turn requires provision of large-scale data sets on leaf and root R. Recent studies reporting on expanded global data sets of leaf Rdark and its T-dependence [11,12,13]—compiled over several years using slow, low-throughput gas exchange protocols—are a step forward. Addressing the need for new, large-scale datasets on plant R will require development of rapid, high-throughput methods capable of overcoming current bottlenecks in data provision

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Conclusion