Scafaro Andrew P, Negrini A Clarissa A, O'Leary Brendan, Rashid F Azzahra Ahmad, Hayes Lucy, Fan Yuzhen, Zhang You, Chochois Vincent, Badger Murray R, Millar A Harvey, Atkin Owen K
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.
Plant Methods. 2017 Mar 21;13:16. doi: 10.1186/s13007-017-0169-3. eCollection 2017.
Mitochondrial respiration in the dark () is a critical plant physiological process, and hence a reliable, efficient and high-throughput method of measuring variation in rates of is essential for agronomic and ecological studies. However, currently methods used to measure in plant tissues are typically low throughput. We assessed a high-throughput automated fluorophore system of detecting multiple O consumption rates. The fluorophore technique was compared with O-electrodes, infrared gas analysers (IRGA), and membrane inlet mass spectrometry, to determine accuracy and speed of detecting respiratory fluxes.
The high-throughput fluorophore system provided stable measurements of 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 in 138 genotypes of wheat; and, (2) quantification of rarely-assessed whole-plant through dissection and simultaneous measurements of above- and below-ground organs.
Variation in absolute 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 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 on multiple samples simultaneously, irrespective of plant or tissue type.
黑暗中的线粒体呼吸是植物的关键生理过程，因此，一种可靠、高效且高通量的测量线粒体呼吸速率变化的方法对于农学和生态学研究至关重要。然而，目前用于测量植物组织中线粒体呼吸的方法通常通量较低。我们评估了一种用于检测多种氧气消耗速率的高通量自动化荧光团系统。将荧光团技术与氧电极、红外气体分析仪（IRGA）和膜进样质谱法进行比较，以确定检测呼吸通量的准确性和速度。
高通量荧光团系统在数小时内对离体叶片和根组织中的线粒体呼吸提供了稳定的测量结果。高通量潜力明显，因为荧光团系统每个样品测量比其他传统方法快10至26倍。该技术的多功能性体现在它能够：（1）快速筛选138个小麦基因型中的线粒体呼吸；以及（2）通过解剖和同时测量地上和地下器官来量化很少评估的全株线粒体呼吸。
不同技术之间观察到绝对线粒体呼吸速率的差异，可能是由于样品条件的差异（即液相与气相、开放与封闭系统），这表明使用不同测量仪器的研究之间进行比较可能不可行。然而，我们提出的高通量方案提供的线粒体呼吸速率值与植物科学家目前最常用的IRGA仪器相似。再加上样品处理速度提高了十倍以上，我们得出结论，高通量方案能够同时对多个样品进行可靠、稳定和可重复的线粒体呼吸测量，而与植物或组织类型无关。
