The combination of gas-phase fluorophore technology and automation to enable high-throughput analysis of plant respiration.
BACKGROUND
Mitochondrial 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 ofRdarkis essential for agronomic and ecological studies. However, currently methods used to measureRdarkin plant tissues are typically low throughput. We assessed a high-throughput automated fluorophore system of detecting multiple O2consumption 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 ofRdarkin 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 ofRdarkin 138 genotypes of wheat; and, (2) quantification of rarely-assessed whole-plantRdarkthrough dissection and simultaneous measurements of above- and below-ground organs.


CONCLUSIONS
Variation in absoluteRdarkwas 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 ofRdarkto 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 ofRdarkon multiple samples simultaneously, irrespective of plant or tissue type.
ARC Centre of Excellence in Plant Energy Biology, Research School of Biology, Building 134, The Australian National University, Canberra, ACT 2601 Australia.|Bayer CropScience SA-NV, Technologiepark 38, 9052 Gent (Zwijnaarde), Belgium.
Andrew P
AP
Scafaro
ARC Centre of Excellence in Plant Energy Biology, Research School of Biology, Building 134, The Australian National University, Canberra, ACT 2601 Australia.
A Clarissa A
ACA
Negrini
ARC Centre of Excellence in Plant Energy Biology, Research School of Biology, Building 134, The Australian National University, Canberra, ACT 2601 Australia.|Australian Research Council Centre of Excellence in Plant Energy Biology, University of Western Australia, 35 Stirling Highway, Crawley, WA 6009 Australia.
Brendan
B
O'Leary
ARC Centre of Excellence in Plant Energy Biology, Research School of Biology, Building 134, The Australian National University, Canberra, ACT 2601 Australia.
F Azzahra Ahmad
FAA
Rashid
ARC Centre of Excellence in Plant Energy Biology, Research School of Biology, Building 134, The Australian National University, Canberra, ACT 2601 Australia.
Lucy
L
Hayes
ARC Centre of Excellence in Plant Energy Biology, Research School of Biology, Building 134, The Australian National University, Canberra, ACT 2601 Australia.
Yuzhen
Y
Fan
ARC Centre of Excellence in Plant Energy Biology, Research School of Biology, Building 134, The Australian National University, Canberra, ACT 2601 Australia.
You
Y
Zhang
ARC Centre of Excellence for Translational Photosynthesis, Building 134, The Australian National University, Canberra, ACT 2601 Australia.
Vincent
V
Chochois
ARC Centre of Excellence for Translational Photosynthesis, Building 134, The Australian National University, Canberra, ACT 2601 Australia.
Murray R
MR
Badger
Australian Research Council Centre of Excellence in Plant Energy Biology, University of Western Australia, 35 Stirling Highway, Crawley, WA 6009 Australia.
A Harvey
AH
Millar
ARC Centre of Excellence in Plant Energy Biology, Research School of Biology, Building 134, The Australian National University, Canberra, ACT 2601 Australia.
Owen K
OK
Atkin
Dark respiration; Fluorophore; Gas-exchange; High-throughput; Oxygen consumption; Oxygen electrodes; Respiration; Respiratory flux; Respiratory quotient