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

Abstract

BackgroundMitochondrial 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.ResultsThe 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.DiscussionVariation 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 materialThe online version of this article (doi:10.1186/s13007-017-0169-3) contains supplementary material, which is available to authorized users.

Predicting dark respiration rates of wheat leaves from hyperspectral reflectance

“…Experiment 1 was carried out at the ANU Controlled Environment Facilities, Canberra, Australia. Three wheat genotypes, “Calingiri,” “Halberd,” and “Janz,” were selected to represent a wide range of average rates of R dark ; an earlier study screening 138 lines (grown in controlled environment cabinets) showed twofold genotypic variation in R dark among the wheat lines, with Calingiri, Halberd, and Janz being at high (0.79 μmol O 2 m −2 s −1 ), mid (0.50 μmol O 2 m −2 s −1 ), and low (0.35 μmol O 2 m −2 s −1 ) range of R dark values, respectively (Scafaro et al, ). Seeds were germinated on moist filter papers on March 9, 2016, with >95% germination achieved within 2 days.…”

“…The dissected leaf section was weighed and exact area determined. The ~4 cm 2 leaf sections were placed in an automated Q2 O 2 ‐sensor (Astec Global, Maarssen, The Netherlands) to determine O 2 consumption rate following the method of Scafaro et al (). Briefly, freshly dissected leaf tissues were placed in 2 ml tubes and hermetically sealed with specialized caps (Astec Global).…”

“…The Q2 O 2 ‐sensor was set at 25°C and measurements taken at a frequency of 4 min over a 2 hr period. However, values from the first 30 min were disregarded, as they tend to be unstable—respiratory activity rapidly increased and decreased during this period (Scafaro et al, ).…”

Variation in Leaf Respiration Rates at Night Correlates with Carbohydrate and Amino Acid Supply

“…Standards containing normal air and 100% N 2 were used to calibrate to 100% and 0% atmospheric oxygen. The oxygen partial pressure was determined to be 20.95% atmospheric pressure, and the ideal gas law was used to calculate molar oxygen consumption rates (Scafaro et al, 2017).…”

“…Although providing robust measurements of respiration, these procedures are not high throughput, and this has limited the scope of experiments aimed at better understanding plant respiration. Recently, fluorometric oxygen sensors have been utilized in multiplexed experiments to measure changes in oxygen concentration in solution (Sew et al, 2013) and in gas phase (Scafaro et al, 2017). Here, our study used high-throughput fluorometric measurements to perform large-scale surveys of leaf R N among Arabidopsis accession populations grown under a common favorable environment.…”