The combination of gas-phase fluorophore technology and automation to enable high-throughput analysis of plant respiration
© The Author(s) 2017
Received: 8 December 2016
Accepted: 17 March 2017
Published: 21 March 2017
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.
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.
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.
Mitochondrial respiration (R) is an essential physiological process in plants required for most energy-dependent metabolic processes. In mature leaves, R takes place in darkness (R dark) and in the light, and is central to processing of carbon assimilates and nitrogen assimilation , while also supporting the energy requirements of phloem loading and maintenance processes (e.g. protein turnover and membrane transport) [2–6]. Respiration is also central to the functioning of roots, providing the energy needed for biosynthesis, nutrient uptake and assimilation, as well as maintenance processes . As such, genotypic and/or environmentally-induced variations in leaf and root R play a crucial role in determining growth/survival of individual plants, and productivity/functioning of terrestrial ecosystems [8–10]. Because of this, 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 R dark and its T-dependence [11–13]—compiled over several years using slow, low-throughput gas exchange protocols—are a step forward. However, our understanding of fine-scale temporal, spatial and developmental variation in plant R remains limited, both for natural and managed ecosystems. 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.
One area where there is an urgent need for data on plant R is within the agriculture industry, where more energy-efficient crops are needed to improve global food security. For wheat (Triticum aestivum), only 10–15% of photosynthetic carbon gain contributes to yield , demonstrating the untapped potential for improving energy use efficiency. 30–80% of daily carbon gain by photosynthesis is subsequently respired [15–18], with respiratory costs increasing with increasing temperature . Given that the efficiency of ATP synthesis per unit of CO2 or O2 equivalents respired varies (reflecting engagement of phosphorylating and non-phosphorylating pathways of mitochondrial electron transport [20, 21]), there is potential to improve crop yields via selecting for efficient genotypes with reduced rates of R [22, 23]. Indeed, there is growing evidence that physiological screening on a large scale assists crop breeders in identifying beneficial genetic material . However, recombinant inbred line (RIL) populations, diversity panels and/or the structured genetic populations used in genome wide association studies (GWAS) typically include many hundreds of plant variants. Studying these for respiratory traits will require thousands of respiratory measurements to be routinely made on material at the same time of day and developmental stage.
Comprehensive R datasets are also needed to improve modelling of respiratory fluxes in terrestrial ecosystems [9, 25–27]. Using standard leaf gas exchange methods, recent surveys have greatly increased our understanding of biome-to-biome variation in leaf R dark [11–13]; our understanding of how sustained changes in the environment affect respiratory rates is also improving [11, 28–31]. Yet, limitations in available data (e.g. documenting environmental, developmental and/or temporal variations) restrict our ability to fully describe the complexity of plant R that occurs in nature. Similarly, respiratory measurements have been conducted in only a small fraction of extant terrestrial plant species, limiting our ability to explore evolutionary changes in plant energy use efficiency. Addressing these challenges requires development of high-throughput methods for quantifying respiratory fluxes of plants growing in natural ecosystem across the globe.
Measurement times required per sample for each of the R dark techniques assessed
Purge tubes of air using N2 gas or sodium dithionite
Dissect tissue (e.g. scalpel, scissors or leaf punch) and place in measuring tube
In general, slopes taken from 1 to 2.5-h. 186 samples per run. Note: more than 186 samples can be simultaneously measured but cycle time between O2 recordings will increase to >6-min, reducing resolution
Prepare and assemble electrodes, including application of membrane and electrode solution. Aerate calibration solutions and obtain zero and saturated O2 values after stabilisation of current
Dissect tissue and place inside cuvette and adjust plunger being careful not to introduce air pockets
Slopes taken after stabilisation of signal and before depletion of O2, usually within 10–40 min but dependent on sample
Change consumables (e.g. soda lime, desiccant, CO2 canister) and zero IRGA chambers
Select and clip measuring chamber onto leaf
Allow steady-state gas-exchange to be reached
Apply membrane and test membrane stability. Purge tube and inject known volumes of O2 and CO2. Record background consumption
Dissect tissue and place inside cuvette and air-seal cuvette
Allow signal to stabilise (usually 5 min) and record slope between 5 and 20 min
Infrared gas-analysers (IRGA) are also commonly used to measure rates of plant R (as respiratory CO2 efflux), exploiting the infrared absorption properties of CO2. The major benefit of the IRGA systems is that they can be portable and operate as a gas-phase/open system. Such systems have been extensively used in recent times for quantifying plant R dark [12, 40–42], including specialised chambers for whole-plant R dark [16, 19, 43]. While a few research teams have developed multiplex systems for single IRGA measurement of four to 12 samples [e.g. 44], most IRGA measurements are made individually, each requiring 10–20 min per sample (Table 1). Consequently, existing IRGA methods are unlikely to provide the high throughput capacity needed to screen for genetic variations in energy use efficiency and/or improved modelling of ecosystem gas exchange.
Less employed spectroscopy technology for detecting respiratory O2 and/or CO2 exchange include tuneable diode laser (TDL) spectroscopy  and cavity ring-down (CRDS) spectroscopy . Mass spectrometry can also be used, with one example of a mass spectrometry technique being membrane inlet mass spectrometry (MIMS), a gas phase method that is used to discriminate between O2 and CO2 isotopes, enabling deeper insight into the photosynthesis/respiratory process [44, 47]. Although MIMS is beneficial in that it can discern gas isotopes, neither it nor the above spectroscopic approaches are high-throughput (Table 1). Similarly, calorimetry measurements of metabolic heat rate and respiratory fluxes [48, 49] while providing an opportunity to explore relationships between respiration and growth—are also not high throughput.
Using O2-sensitive fluorophores in combination with fibre-optic fluorescent detection mechanisms for measuring the O2 evolution of photosynthesis of illuminated leaf disks was occurring by the late 1990s . The technique works by exciting a fluorophore, in most cases a metal porphyrin, whose fluorescence is sensitive to O2 quenching. The measured decay rate of the fluorescent emission is thus proportional to the partial pressure of O2 present [51, 52]. This technology is becoming a more common technique for detecting respiratory O2 consumption of biological samples ranging from bacterial plankton to benthic meiofauna [53, 54]. The power of this technology is that many tissue types of varying abundance can be simultaneously and accurately measured. For example, fluorophore technology has enabled multiple simultaneous measurements of leaf, root and seed respiratory rates . The authors highlight the high-throughput and small tissue size capabilities of the technique, not achievable using conventional Clark-type electrodes, infrared gas-analyser, spectroscopy or calorimetry methods. Yet, take-up of fluorophore technology to facilitate high-throughput measures of plant R remains limited, reflecting the need for more straightforward sample preparation than was possible using the liquid-phase approach of Sew et al. . By contrast, using fluorophore technology in a gas-phase medium is likely to lead to faster processing times and avoid technical issues, such as floating tissues and air-pockets. To date, automated gas-phase measurements of O2 consumption using fluorophore techniques for plants have primarily focused on large-scale analysis of seed germination [56, 57], with automated, high-throughput assessments of non-seed plant R yet to be attempted using gas-phase fluorophore approaches.
To address the urgent need for high-throughput measurements of plant R dark, we have trialled an approach for measuring respiratory O2 uptake which re-purposes equipment designed for seed germination assays and combines the advantages of: (1) fluorophore technology that can accurately measure changes in O2 partial pressure in small measuring volumes that are easily calibrated; (2) closed, gas-phase measurements, which require minimal preparation time; and, (3) an automated sampling mechanism, relying on robotics to take measurements of multiple samples within a short period of time. As part of our study, we compare multiple O2 consumption detection methodologies to ascertain the reliability and compatibility of the different approaches. Further, to illustrate the potential of the high-throughput fluorophore technology to accelerate our understanding of plant R dark, we report on: (1) a screen of R dark in 138 genotypes of wheat (using >550 plants) 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.
The species used in this study were a grass (wheat—Triticum aestivum), a herb (thale cress—Arabidopsis thaliana) and an evergreen broadleaved tree (red river gum—Eucalyptus camaldulensis), enabling the method to be tested on a range of plant functional types. Considering its agricultural significance, T. aestivum was selected as the primary species of interest, and all experiments, including the high throughput practical applications, were undertaken on T. aestivum, with a sub-set of other experiments conducted using other tissues. All experiments took place at the Research School of Biology at the ANU, Canberra, Australia plants grown in organic potting mix, enriched with Osmocote® OSEX34 EXACT slow-release fertiliser, following manufacturer’s instructions (Scotts Australia, Bella Vista, NSW) with an N/P/K ratio of 16:3.9:10. Plants were watered daily to field capacity. For experiments where roots were analysed, wheat plants were grown hydroponically in a nutrient solution consisting of 1.4 mM NH4NO3, 0.6 mM NaH2PO4·2H2O, 0.5 mM K2SO4, 0.2 mM CaCl2·2H2O, 0.8 mM MgSO4·7H2O, 0.07 mM Fe-EDTA, 0.037 mM H3BO3, 0.009 mM MnCl2·4H2O, 0.00075 mM ZnCl2·7H2O, 0.0003 mM CuSO4·5H2O, 0.0001 mM (NH4)6Mo7O24·4H2O, 0.000138 mM NH4VO3, and 0.0012963 mM Na2SiO3. A pH ranging from 5 to 6 was maintained by adding concentrated sulphuric acid or sodium hydroxide, and monitoring of pH using a portable pH meter (Rowe Scientific Pty. Ltd., NSW, Australia). The hydroponic solution was aerated continuously using Infinity AP-950 aquatic air pumps (Kong’s Pty Ltd, Ingleburn, Australia). Plants were grown at temperatures of 25/20 °C for T. aestivum and E. camaldulensis, in temperature controlled greenhouses with natural photosynthetically active radiation (PAR) of between 400 and 1200 μmol m−2 s−1. A. thaliana was grown at 22/15 °C in temperature-controlled growth chambers (Thermoline, Wetherhill Park, Australia) with a PAR of 200 ± 30 μmol m−2 s−1 and a 12:12 h light/dark photoperiod. For leaf dissection samples, broad-leaved A. thaliana and E. camaldulensis leaf tissue was extracted using brass coring tools of known diameter and for T. aestivum a set distance of leaf blade was dissected with a scalpel. Where sectioned, root segments were dissected transversely from base to tip.
High throughput fluorophore measurements
A Q2 O2-sensor (Astec Global, Maarssen, The Netherlands) designed and marketed for seed germination assays was used to obtain automated, high-throughput fluorophore measurements of dark respiration from plant material. A custom-built frame covered in black cloth was used to maintain darkness during sample measurements. Plant material were freshly dissected and placed in empty tubes (1, 2 or 4 ml in volume) and hermetically sealed with specialised caps (Astec Global). The top surface of caps contained a fluorescent metal organic dye, sensitive to O2 quenching. A blue-spectrum LED excitation pulse (approximately 480 nm) onto the surface of caps, followed by emission detection in the red spectrum (approximately 580 nm), enables the O2 dependent decay in fluorescence signal to be quantified. The fibre optic fluorescence detection unit is attached to a robotic arm which sequentially measures vials placed in racks of 48 tubes each (or 24, 4 ml tubes). The machine can accommodate 16 racks allowing 768 samples (1 or 2 ml tubes) to be measured in a single run. The frequency of measurements was in most cases set to 4 min, enough time to measure approximately 180 samples (a minimum measurement frequency of 1-min is required). The Q2 O2-sensor is calibrated before each set of measurements by measuring a designated tube containing ambient air (designated 100% O2), and a tube purged of all O2 using a sodium dithionite solution, or alternatively purging the tube of air using N2 gas (designated 0% O2). Output is given as an O2 percentage, relative to the calibration readings.
Respiratory consumption of O2 by leaves (3–42 mg fresh mass) or roots (56–214 mg fresh mass) were measured in the liquid-phase using Oxytherm Clark-type O2-electrode (Hansatech Instruments, Pentney, UK) in a 2 ml measuring volume. Electrodes were calibrated by bubbling water with compressed air for approximately 2-h to reach saturation followed by adding sodium dithionite to record O2 depleted signals. Leaf and root respiration was measured in a solution containing 20 mM Hepes (pH 7.2), 10 mM MES and 2 mM CaCl2, at 21.5 ± 1 °C. All measurements were made by dark adapting tissue for >30 min, submerging tissue in the Clark-type electrode cuvettes below measuring solution, with no obvious air pockets and continually stirring, and recording O2 consumption using Oxygraph Plus v1.02 software (Hansatech Instruments). The linear part of O2 consumption (approximately 10–30 min into each run) was used to calculate respiration rates.
Infrared gas-analysis of CO2 efflux by respiring leaves was measured using a Licor 6400XT with a 3 × 2 cm chamber head ((LI-COR, Lincoln, Nebraska, USA) on >30 min dark-adapted leaves. Attached whole leaves were placed across the measuring chamber and chamber gaskets and measurements recorded after CO2 readings stabilised (~10–15 min). The flow rate was set to 300 µmol s−1, the block temperature set to ambient air temperature of 22 °C and the CO2 reference sample was set to 400 µmol mol−1, to match ambient air. The light source was turned off.
Membrane inlet mass spectrometry
Dark-adapted wheat leaf disks (3 × 0.5 cm2 or 6 × 0.5 cm2) were placed in a 1 mL O-ring sealed cuvette containing only air and a polyethylene membrane sealed outlet attached to a mass spectrometer (MM6: VG, Winsford, UK). O2 (m/z = 32) and CO2 (m/z = 44) detection over a 20-min period was recorded. Prior to leaf disk samples being placed in the cuvette, N2 gas purging of the cuvette and injections of known volumes of O2 and CO2 allowed for conversion of mass detection signal to a gas concentration and the background consumption rate of O2 and CO2 by the mass spectrometer to be accounted for when determining leaf derived O2 consumption and CO2 evolution rates.
Replication and statistical analysis
For all experiments four to six biological replicates, with a biological replicate considered as plant material from individual plants grown in separate pots, or containers (when grown hydroponically) were measured. For the comparison of respiratory techniques, two or more samples from each biological replicate were analysed by each technique and sampling was standardised by selecting a 2 cm long mid-section of young, healthy, fully expanded leaves, or in relation to root samples, a longitudinal section from base to tip of the longest root segment. A one-way ANOVA was used to determine significance between leaf O2 consumption techniques and Two-Sample t-tests for differences between leaf CO2 evolution techniques and root O2 uptake techniques.
Technical and biological reliability and accuracy
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). The decline was linear in all species and tissues tested. The 90-min O2 consumption slope between 1 and 2.5-h had a mean r 2 of 0.99 across both species (Fig. 1b). Typically, the initial 0–30 min period of each run was associated with sharp declines in the O2 consumption slope. R dark calculated from a 1-h moving average of slopes over 7-h was similar to the slope of O2 consumption over a set 90 min period between 1 and 2.5-h (presented as dashed horizontal lines in Fig. 1c). The O2 consumption slope between 1 and 2.5-h can therefore be used as a standard period for calculating R dark across experiments.
Although increased CO2 concentration was not inhibitory to R dark, heavy mechanical wounding of tissue resulted in higher R dark (Fig. 2c). Intact wheat leaves versus a 2 × 0.5 cm transverse section from the middle of leaves (a ratio of 1:1, wounded boundary length to leaf area) did not exhibit significant differences in R dark on an area basis (Fig. 2c). However, if the transverse section was further sliced into 20 smaller pieces (a 20-fold increase in the cut surface length to leaf area ratio), R dark increased by as much as two-fold (Fig. 2c). Applying a buffered saline solution to the heavily wounded leaf partly mitigated the enhancement of R dark by wounding. Thus it is important to reduce the amount of tissue exposed to mechanical damage when processing samples, to avoid the risk of artificially enhancing respiration rates.
Comparisons between leaf gas-exchange methods
Considering the many methods currently in use for determining plant respiratory gas-exchange, and the need to ensure that the fluorophore system was giving comparable rates, we compared R dark values generated using the fluorophore technology, the more conventional Clark-type O2-electrodes, Licor 6400 IRGA gas-exchange system, and membrane inlet mass spectrometry (MIMS). All of these techniques have varying degrees of difference in sample preparation and technical methodology that may influence the final respiratory rate recorded. For example, while we measured O2 consumption in the gas-phase using the fluorophore technique, O2-electrode measurements were made in aqueous-phase. Despite the IRGA measurements being made in gas-phase, measurements were of CO2 rather than O2 flux, and in an open gas-exchange system rather than the closed fluorophore system. Furthermore, IRGA measurements are made on intact not detached leaves. MIMS would be closest in methodology to the fluorophore technique in that both were measuring in the gas phase, in an essentially closed system. However, the MIMS system is not a completely closed system as the gradual leak of gasses through the semi-permeable membrane to the mass spectrometer would lead to changes in partial pressure and water vapour at the site of the leaf.
High-throughput analysis of respiration
Two studies were undertaken to verify the capabilities and versatility of automated O2 fluorophore technology for measuring high-throughput plant respiration in leaves and other plant tissues.
With the potential to run a single sample using the fluorophore system in less than 2 min (Table 1), a single replicate of all 138 genotypes could be processed in less than 4 h, and potentially, a fully replicated 138 genotype study could be achieved in a single day. The number of samples per day is limited by the capacity of the robotic system, and by the time taken to prepare samples. By comparison, the other techniques have significantly longer calibration, sampling and measurement times required to acquire a single measurement (Table 1). Hence, what can be undertaken in 8-h using the high-throughput fluorophore technique, would require a minimum of 83 equivalent hours, or as much as 200-h for other commonly used procedures to measure R.
We demonstrate that using robotic fluorophore-based gas-phase measurements of O2 consumption in sealed tubes provides a simple yet reliable and reproducible means of measuring R dark for a diverse range of plant tissue types and species. The technique differentiates itself from other conventional methods in that it significantly reduces the time required for sample preparation and has substantial simultaneous measuring capabilities, making the technique a truly high-throughput means for measuring respiration. We demonstrate the potential capabilities of the method by measuring R dark of 138 wheat genotypes, and by measuring R dark of all tissues of six mid-vegetative stage plants simultaneously. A comparison of R dark in absolute terms, generated by different methodologies suggests variation in respiratory rates depending on technique employed, which should be considered when making direct comparisons between methods.
Strengths and weaknesses of high-throughput fluorophore methods
There was an initial spike and rapid decline in respiratory activity within the first 30-min of measurements (Fig. 1b). We dark-adapted leaves for a minimum or 30-min prior to fluorophore analysis, so although it is common to find a spike in respiration of leaves following exposure to light within the initial 30-min post-illumination period , post-illumination bursts in respiration do not explain the findings. Furthermore, while the O2-electrode and MIMS measurements continuously recorded in a similar manner to the fluorophore system, neither approach showed the initial spike, followed by rapid decline in R dark that was exhibited by the fluorophore approach (Fig. S3). Consequently, the first 60-min of each run were not used to calculate rates of R dark in the genotypic and developmental studies; the initial stabilisation period, however, can be used as a dark-adaptation period if tissue is not dark-adapted prior to fluorophore experimentation.
CO2 has previously been postulated to inhibit cytochrome c oxidase (COX) activity . Reports initially suggested that a doubling of current atmospheric CO2 (i.e. from 0.04% of atmospheric gas to 0.08%) reduced R dark by 15–30% [60–62]. However, it was later discovered that CO2 inhibition of R dark was mostly likely an artefact of the measuring techniques used to quantify respiratory CO2 release [63–65]. Our results show that CO2 accumulation does not inhibit R dark. In fact, even with CO2 concentrations surrounding the sampled tissue reaching more than 90% of the gas volume (a 450-fold increase in concentration relative to previously reported measurements), no substantial inhibition in respiration occurred (Fig. 2b). We therefore conclude that leaf R dark is highly insensitive to CO2 accumulation over a course of several hours.
One factor that does seem to influence R dark is mechanical wounding (Fig. 2c). Leaf wounding was thought to affect leaf respiration as far back as 1950 . Increased R dark with mechanical wounding is attributed to stimulation of the ATP/ADP ratio and activation of pyruvate kinase due to ion changes associated with wounding . Pre-treatment by washing leaf samples with a buffered saline solution, the same as the measuring solution in liquid phase measurements, reduces any wounding effects on leaf R [38, 68]. We observed an increase in R dark when a large proportion of the sample had a wounded edge, and a reduction in R dark by applying wounding buffer, although not enough of a reduction to eliminate the wounded effect (Fig. 2c). However, minimal wounding did not significantly change R dark. Considering the time required to wash the sample tissue with a wounding solution, we suggest minimising as much as possible the mechanical wounding of tissue, rather than applying a wounding solution, if high-throughput sampling is desired. However, minimising mechanical wounding may require using larger volume tubes (e.g. moving from 1 to 4 mL tubes) to adequately fit sample tissue. By running a preliminary experiment, one could initially check for wounding effects and use the appropriate tissue size thereon after.
The limited effect of leaf wounding and lack of any inhibition to R dark from CO2 accumulation resulted in respiration measurements being stable over a period of many hours (Fig. 1). The stability of R dark for small leaf sections means that although the fluorophore technique we present is a closed-system that destroys the sampled tissue, a small sample of leaf collected in the field can be transported to the lab (making sure to keep detached leaves from desiccating), accurately representing in situ R dark. Thus, the fluorophore method can be considered as a pseudo non-destructive technique for high-throughput analysis for field experiments, as demonstrated below in the 138 wheat genotypes study we present.
Comparisons between respiratory methods
Although Hunt  comprehensively compared the strengths and weaknesses of multiple photosynthesis and respiration measurement techniques, no study to our knowledge has directly compared the absolute values of R obtained from the same biological material but measured across multiple techniques. Determining if the fluorophore technique presented in our study is comparable with previously well-established methods is important. Firstly, if results are to be examined among studies that utilised different techniques, it must be established if the analysis is viable, or whether differences among studies are an artefact of measuring technique. Secondly, although in many cases only the relative differences in R between samples may be of interest (for example, the genotypic study we present here), in many circumstances, absolute R will be desired, such as for determining absolute photosynthesis, or modelling the impact of R on terrestrial carbon budgets. Hence, we directly compared fluorophore, O2-electrode, IRGA, and MIMS output (Fig. 3). We found differences did exist between the techniques, suggesting that comparing results between studies utilising different R measuring apparatus may not be appropriate, or at least with the caveat that comparisons may require cross-calibration of method. Differences in measurements based on either O2 consumption or CO2 evolution may be expected considering the respiratory quotient (RQ) will not necessarily be equal to 1 (i.e. respiratory CO2 release being equal to O2 uptake) if pure carbohydrates were not the only source of respiratory substrate, or the oxidation state of respiratory products differed, although a RQ of 1 is usually assumed for higher plants under non-stressed conditions . Indeed, the simultaneous measurement of R dark derived from O2 and CO2 exchange by MIMS gave close to matching values, supporting a RQ of 1, in contrast to a study of wheat leaves measured in the dark, 6-h into the light period (similar conditions to this study), which gave a RQ value of 1.8 ± 0.21 . However, the study by Azcón-Bieto, Lambers and Day  used values of R determined separately using O2-electrode and IRGA systems, and since we found lower O2 based O2-electrode values relative to CO2 IRGA values, we emphasise that caution must be taken when comparing R calculated from different methodologies. Of note, the widely used IRGA gas-exchange system on intact leaves gave similar rates to the fluorophore results, suggesting the two techniques may be complementary. We did not undertake subsequent experiments to determine the specific reasons for variations in R dark between the techniques compared, and it will be of interest to further explore the reasons for why the techniques vary in future studies.
Genotypic and whole-plant analysis
Both a comprehensive genotype comparison and whole-plant respiratory balances were successfully obtained by use of the gas-phase automated fluorophore technique. Interestingly, a more than two-fold variation in R was observed between the 138 wheat genotypes (Fig. 4a). This demonstrates the inherent intra-specific divergence of R in Triticum aestivum, and a potential target for future yield improvements, if R not contributing to growth or yield can be minimised. Inherent differences in R dark between species populations have previously been noted, such as in the ryegrass species Lolium perenne, attributed to adenylate limitations on glycolysis and varying ATP turnover rates between populations . R was also highly variable among genotypes. This may not be considered surprising as leaf functional traits vary considerably among populations/genotypes within a given species. For example, a study of 13 common alpine species found that 30% of observable variance in measured traits, such as specific leaf area and leaf nitrogen content, was among populations/genotypes of a given species . Similar results were found for species growing in a dry tropical forest . Considering R is highly variable among genotypes within species, to gain sufficient statistical power a high level of replication is required (Fig. 4b, c), further supporting the benefit of the high-throughput fluorophore technique we present.
Our whole-plant respiratory analysis demonstrated the important effects of plant development on leaf R and partitioning of R between tissue types, as previously demonstrated in Arabidopsis by Sew et al. , which could be detrimentally ignored if the power of high-throughput respiratory analysis was not readily available. The results highlight the fact that, when measuring leaf, stem and root O2 uptake in the gas phase, leaf R dark accounted for 51% of the entire R budget. In other words, close to half of all vegetative-stage wheat R occurs in non-leaf tissue, a finding reported for previous studies that quantified whole-plant CO2 fluxes [15–19]. Yet, we tentatively suggest that the majority of plant R reports would focus entirely, or predominantly on leaf R. Furthermore, the oldest and newest emerging leaves had considerably higher mass-based rates of R dark than intermediate aged leaves. In regards to the latter, this is presumably due to the added cost of growth R as well as maintenance R for newly emerging leaves . The spike in R for the oldest leaves may reflect the costs associated with senescence, such as an energy expensive remobilisation of nutrients from the senescing leaf to other parts of the plant. For example, in oats (Avena sativa), promotion of senescence of leaves by withholding light leads to a greater than two-fold increase in O2 consumption, attributed to decoupling of R dark from oxidative phosphorylation, and amino-acid and soluble sugar liberation during senescence .
The high-throughput and tissue size versatility of the experiments we conducted highlight the comparative advantages of an automated gas-phase system, over other systems based on the same technology but reliant on aqueous-phase and limited sample tubes and volumes. Although aqueous-phase fluorophore systems may be relatively high-throughput when compared to the older technology of Clark-type O2 electrodes, liquid-phase measurements still require extensive time in preparation of solutions, dispensing of solutions, and delicate sample positioning or sufficient stirring to facilitate O2 movement to the sensor [e.g. 75]. We processed 138 samples, from tissue harvesting to initial O2 uptake measurements, in a period of less than 2-h, which was possible due to the simple procedure of placing tissue in tubes, tightening the caps and placing tubes in the designated instrument position. Such a fast turnaround for sample processing would not be possible in a non-fluorophore and/or aqueous-phase procedure. The speed at which samples can be processed and the versatility in sample size and tissue type enables respiratory analysis that simply would not be feasible using other established approaches. The simultaneous measurement of many genotypes and the construction of multiple whole-plant respiratory budgets emphasise the potential of this method and its wider application.
APS, ACAN, BO, AHM and OKA conceived the idea for the study. APS, ACAN, BO, FAAF, LH, YZ, VC and MRB conducted the experiments. APS wrote the first draft; all authors contributed significantly to subsequent versions. All authors read and approved the final manuscript.
The support of the Australian Research Council (CE140100008) to OKA and AHM is acknowledged.
The authors declare that they have no competing interests.
Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
Australian Research Council (CE140100008).
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