Researchers often want to study the respiratory properties of individual parts of plants in response to a range of treatments. Arabidopsis is an obvious model for this work; however, because of its size, it represents a challenge for gas exchange measurements of respiration.
The combination of micro-respiratory technologies with multiplex assays has the potential to bridge this gap, and make measurements possible in this model plant species. We show the adaptation of the commercial technology used for mammalian cell respiration analysis to study three critical tissues of interest: leaf sections, root tips and seeds.
The measurement of respiration in single leaf discs has allowed the age dependence of the respiration rate in Arabidopsis leaves across the rosette to be observed. The oxygen consumption of single root tips from plate-grown seedlings shows the enhanced respiration of root tips and their time-dependent susceptibility to salinity. The monitoring of single Arabidopsis seeds shows the kinetics of respiration over 48 h post-imbibition, and the effect of the phytohormones gibberellic acid (GA3) and abscisic acid (ABA) on respiration during seed germination.
These studies highlight the potential for multiplexed micro-respiratory assays to study oxygen consumption in Arabidopsis tissues, and open up new possibilities to screen and study mutants and to identify differences in ecotypes or populations of different plant species.
Plant cells rely on mitochondrial respiration for ATP, carbon skeletons for amino acid assimilation and organic acid building blocks for biosynthetic pathways. Respiration is the principal component in CO2 loss from cells and is a key factor in the assessment of the carbon balance of plants and in defining the factors influencing the plant growth rate (Amthor, 1989). The assessment of the cellular respiration rate therefore provides an important insight into the metabolic activity and physiological state of plant tissues (Lambers, 1985). The respiration rate can be measured noninvasively as gas exchange from the surface of tissues via the monitoring of the rate of O2 consumption or CO2 production. O2 consumption measurements have relied on low-throughput and time-consuming gas- or liquid-phase analysis of O2 concentration by polarographic Clark-type oxygen electrodes in closed systems (Walker, 1990; Hunt, 2003). CO2 production has been measured using gas-phase infra-red gas analysers in closed systems or in differential open system configurations (Hill & Powell, 1968; Hunt, 2003). Micro-electrodes based on polarographic methods have also been used to monitor O2 concentrations inside seeds and siliques (Porterfield et al., 1999) and in root tissues (Armstrong et al., 2000). Recently micro-electrodes have even been adapted to measure respiration inside single photosynthetic cells (Bai et al., 2011). However, these miniaturized methods are highly technical, low throughput, require substantial specialization and often involve painstaking adaptation for use on specific tissues of the target plant species.
Arabidopsis has now become the key model for understanding the molecular components of respiration in plants. Most of our recent advances in the understanding of the biogenesis of mitochondria and the retrograde regulation of respiration by intracellular signalling processes has originated from studies in this species (Millar et al., 2011). However, reports of the measurement of the respiration rate of Arabidopsis, and how it is altered when mitochondrial functions are changed, have been limited as a result of two key constraints. First, the small size of many Arabidopsis tissues has limited the options for the use of many conventional gas exchange systems to measure respiration rates (and micro-respirometry, such as that reported in Arabidopsis siliques (Porterfield et al., 1999), is a very specialized field). Second, the lack of high-throughput assay systems has limited the full use of the resources in Arabidopsis biology to assess respiratory phenotypes through the access of a wide range of mutants, ecotypes and tissue types.
The development of analyte-selective fluorophores, which monitor the partial pressure of oxygen, coupled to fibre-optic cables to monitor their fluorescent properties, has opened up new opportunities in respiratory measurements. Fluorophore-based micro-oxygen sensors have been used to monitor oxygen levels inside plant seeds (Borisjuk & Rolletschek, 2009; Ast et al., 2012) and in the root rhizosphere (Rudolph et al., 2012) to study hypoxia. These measurements are of oxygen concentration, not respiration rate, and so diffusion of gases for specific tissues needs to be calculated or standardized for respiration rates to be deduced by time series measurements of oxygen concentration (Rudolph et al., 2012). Coupling fluorophore-based micro-oxygen sensors to microtitre plate assays, for which standardized diffusion rates can be calculated, has allowed the high-throughput analysis of the respiration rate in milligrams of tissue in microlitre volumes (Ferrick et al., 2008; Gerencser et al., 2009). Such systems have been commercialized and are now being used to measure cellular respiration rates and cellular bioenergetics of isolated mitochondria and cells from mammalian tissues (Beeson et al., 2010; Rogers et al., 2011; Zhang et al., 2011, 2012). However, to our knowledge, the adaptation and use of such systems for intact plant tissues has not been tested systematically.
Here, we present optimized methods to adapt the use of commercial microplate assays of oxygen consumption by analyte-selective fluorophores to measure the respiration rates of Arabidopsis leaf, root and seed samples. We show that this approach allows high-throughput measurements of the respiration rate in leaf laminar and vascular regions of a single leaf, the respiration rate of single root tips and even the respiration of single imbibed seeds. We illustrate that biological changes in respiration associated with leaf development, leaf age, root segments and hormone-dependent changes in seed germination can be measured and compared. These developments, and the use of commercial systems and consumable packs already optimized and available to researchers, open up opportunities for the in-depth analysis of respiratory phenotypes and their relation to developmental processes in small tissue samples from a variety of plants.
Materials and Methods
Extracellular Flux Analyzer XF96 and 96-well plate set-up
Seahorse XF96 Extracellular Flux Analyzer measurement is based on the fluorimetric detection of O2 levels via fluorophores in a commercial sensor cartridge. Oxygen quenches the fluorescence of a fluorescein complex, the fluorescence is detected by a fibre-optic waveguide and converted into the basal oxygen consumption rate (OCR). During the ‘measurement’ phase, the concentrations are measured continuously until the rate of change is linear, and then OCR is determined from the slope. The probes lift whilst in the ‘mixing’ and ‘waiting’ steps to allow the larger medium above to mix with the medium in the transient micro-chamber, re-oxygenating the solution and thus restoring the oxygen concentration values to baseline. The XF96 data can be visualized and analysed in both XF96 Analyzer software and an Excel-based data viewer. For the underlying calculations, the reader is referred to the literature on the development of this system (Ferrick et al., 2008; Gerencser et al., 2009). Respiration measurements were performed in an XF96 Extracellular Flux Analyzer (Seahorse Bioscience, Billerica, MA, USA) to obtain the OCR of plant tissues. The 96-well sensor cartridge was hydrated in 200 μl per well of XF Calibrant Solution (Seahorse Bioscience) overnight at 37°C before the assay. Several hours before the measurement commenced, the heater of the instrument was turned off to obtain a stable internal measurement temperature in the machine at c. 28°C. Plates (and injection ports when indicated) were filled using multichannel pipettes or en masse by a 96-well robotic liquid handling station (Bravo; Agilent Technologies, Mulgrave, VIC, Australia) using in-house-developed device and protocol programs.
Leaf respiration rate measurements by XF96
Wild-type seeds of Arabidopsis thaliana (L.) Heynh (ecotype Columbia) were placed on wet filter paper and incubated at 4°C for 3 d. The imbibed seeds were transferred to individual pots containing a 1 : 3 perlite : soil mix and covered with a transparent acrylic hood to maintain humidity. The seedlings were grown in a controlled environment growth chamber maintaining a short-day photoperiod (8 h : 16 h, light : dark), a photon flux of 150 μmol photons m−2 s−1, a relative humidity of 75% and a temperature cycle of 22°C : 17°C, day : night temperature regime. When the seedlings were established, the acrylic hood was removed and the plants were subsequently grown with regular watering. At an age of 4–6 wk, as indicated, the plants were used for the measurements.
Single leaf discs were immobilized in wells with either Cell-Tak (BD Bioscience, North Ryde, NSW, Australia) or a commercial skin adhesive Leukosan® (BSN Medical, Mount Waverley, B.C., VIC, Australia) mixed with agarose. For Cell-Tak adhesion, 16 μl of the Cell-Tak mixture, pH 7 (5% (v/v) Cell-Tak, 45 mM sodium bicarbonate, pH 8.0), was used to coat the bottom of each well of the microtitre plate. The absorption of Cell-Tak to the well bottom was allowed for 20 min at room temperature, after which the Cell-Tak mixture was discarded by aspiration before rinsing with distilled water. Single 2.5-mm-diameter leaf discs, which had been freshly cut with a leaf punch, were then placed at the centre of each well and gently pressed to the well bottom using a cotton bud. An even contact between the leaf disc and the Cell-Tak-coated layer on the bottom of the well was required for optimal adhesion. Adhesion was allowed for 30 min before 200 μl of respiration buffer (10 mM HEPES, 10 mM MES and 2 mM CaCl2, pH 7.2 (Atkin et al., 1993; Armstrong et al., 2006a)) was added to the wells. For the skin-glue adhesive and agarose mixture, a combination of 2.5% (v/v) Leukosan® adhesive in 0.25% (w/v) agarose was prepared and kept above 60°C to avoid solidification. For each well, 1 μl of the adhesive mixture was pipetted onto the centre of the well bottom. Then, 2.5-mm-diameter leaf discs were positioned on top of the mixture before gentle pressure with a cotton bud. As the adhesive mixture sets in c. 2 min, sequential handling of the samples is required if large numbers of leaf discs are used. After 2 min, respiration buffer can be added on top of the leaf discs to avoid dehydration. A full plate of 96 leaf discs could be manually adhered in c. 45 min. Once leaf adhesion had been achieved, wells were filled with 200 μl of leaf respiration buffer and loaded into the plate reader after the calibration steps. The time events for both basal respiration measurement and injection were mixing (3 min), waiting (4 min) and measurement (5 min). The method allowed for 10 cycles of mixing, waiting and measurement. The OCR of single leaf discs was recorded by Seahorse XF Acquisition and Analysis Software (Version 1.3; Seahorse Bioscience).
Root respiration measurements by XF96
Seeds of Arabidopsis (A. thaliana) ecotype (Columbia-0) were sown on half-strength Murashige and Skoog (MS) Gamborg B5 plates containing 0.8% (w/v) agar, 1% (w/v) sucrose, 1.8 mM MES at pH 5.8 adjusted by KOH. The plates were placed at 4°C in the dark for 2 d and then transferred to a growth room with a photoperiod of 16 h : 8 h, light : dark at a light intensity of 200 μmol m−2 s−1, relative humidity of 70% and temperature cycle of 22°C : 17°C, day : night. The plates were set in a vertical position. After 7 d of growth, c. 5 mm of the expanded section or elongating root tip were cut for respiration assay with eight replicates for each treatment. The 96-well sensor cartridge was hydrated in 200 μl per well XF Calibrant Solution (Seahorse Bioscience) as mentioned above. After calibration, the 96-well utility plate was filled with 100 μl of respiration buffer containing 0, 100, 200 or 400 mM NaCl. In each well, a single root tip (tip; c. 5 mm) or root expanded section (EXP; c. 5 mm) was added to the bottom of the well. The time events for both basal respiration measurement and injection were mixing (2 min), waiting (3 min) and measurement (5 min). Seven cycles of mixing, waiting and measurement were applied for time course measurements. The OCR of the single root tip or expanded section was recorded by Seahorse XF Acquisition and Analysis Software (Version 1.3; Seahorse Bioscience).
Seed respiration measurements by XF96
For multiple seed measurements, intact seeds (c. 1 mg) were placed in a 96-well plate and surface sterilized by soaking for 7 min in 12.5% (w/v) NaClO and 0.1% (v/v) Tween20, followed by two washing steps with distilled H2O. After this, wells were filled with 200 μl of seed respiration medium (5 mM KH2PO4, 10 mM TES, 10 mM NaCl, 2 mM MgSO4, pH 7.2) and loaded into the plate reader after the calibration steps using the Bravo liquid handling station (Agilent Technologies). Where indicated, inhibitors were added to the medium with a final concentration of 2 μM for KCN or 5 mM for salicylhydroxamic acid (SHAM). Oxygen concentrations before and after inhibitor injection were determined by 11 cycles of mixing (3 min), waiting (4 min) and measurement (5 min). The OCR of seeds was recorded by Seahorse XF Acquisition and Analysis software (Version 1.3; Seahorse Bioscience), and each well was normalized by the milligram weight of seeds used.
For single seed measurements, a sterile solution of 0.25% (w/v) agarose was used, and kept at 65°C to avoid solidification. The agarose solution was pipetted (1 μl) into the centre of each well bottom. Seeds were sterilized by overnight incubation with chlorine gas (100 ml of 12% NaOCl and 3 ml of 37% HClO) in a closed vessel. Each single seed was placed with a sterile toothpick, making sure the adhesion of each seed was in the centre of the well. Then, the wells were filled with 200 μl of seed respiration medium and loaded into the plate reader after the calibration steps. Where indicated, hormones were added to the respiration medium with a final concentration of 2.4 μM for abscisic acid (ABA; PhytoTechnology Laboratories, Shawnee Mission, KS, USA) and 1.2 mM for gibberellic acid (GA3; Sigma-Aldrich). The respiration measurements were made by mixing (3 min), waiting (4 min) and measurement (60 min). The method was run for 48 cycles, achieving a total of 50 h of measurements. The OCR of single seeds was recorded by Seahorse XF Acquisition and Analysis Software (Version 1.3; Seahorse Bioscience).
Leaf respiration by Clark-type oxygen electrode
Plants were grown under the conditions described for XF96 above. The OCR of leaf discs was measured using a liquid-phase Oxygraph system (Hansatech Instruments, Pentney, Norfolk, UK). Before the measurement, the electrode was calibrated at 25°C by the addition of sodium dithionite to 1 ml of aerated autoclaved water to completely deplete oxygen. Leaf discs totalling 40–60 mg fresh weight (FW) of 7-mm-diameter leaf discs were immersed in leaf respiration buffer and incubated in the dark for 30 min. Leaf respiration was performed in a 2-ml volume for at least 15 min at 25°C in a darkened electrode chamber. The amount of oxygen being consumed by the leaf discs was recorded using Oxygraph Plus v1.02 software (Hansatech Instruments), and the OCR (nmol min−1 g−1 FW) was calculated accordingly to the FW of the leaf discs.
The statistical software package IBM SPPS Statistics 19 (IBM Australia, St Leonards, NSW, Australia) was used for data analysis where indicated. An analysis of variance, followed by multiple comparison using post hoc tests and Tukey's honestly significant difference (HSD) mean separation test, was performed to determine the statistical significance of differences of the mean values at P ≤ 0.05.
Adhesion of leaf discs for respiratory measurements
Making OCR measurements in microtitre plates of the Seahorse XF96 requires that the tissues remain at the bottom of the well and do not move during the cycles of mixing and measurement. This requirement is not present when using oxygen electrode or infra-red gas analysis techniques, and is much less of a problem when using mammalian tissues as they are not buoyant structures. To develop suitable adhesion techniques, we trialled two different methods: one using mixtures containing 5% (v/v) Cell-Tak (BD Bioscience) and another using 2.5% (v/v) Leukosan® adhesive in 0.25% (w/v) agarose. Both adhesion methods were found to immobilize leaf discs submerged in buffer for several hours. However, the investigation of the effectiveness of the two adhesion mixtures during the course of the mixing assays showed that the Leukosan® adhesive treatment produced far fewer leaf disc detachment events and a lower standard error for OCR (Supporting Information Fig. S1A,B). Analysis showed that an OCR of 143 ± 11 pmol O2 min−1 (for a leaf disc of c. 0.7 mg FW) could be consistently measured. Replicate leaf discs from the same leaf gave more consistent results than leaf-to-leaf comparisons, suggesting some variability of OCR between leaves (Fig. S1A,B). To test the effect of the adhesive on OCR, we performed similar measurements using 7-mm-diameter leaf discs in a Clark-type oxygen electrode (Oxygraph; Hansatech Instruments). The mean OCR g−1 FW of leaf discs did not change with increasing amount of leaf discs adhered together during the analysis, indicating no substantial effect of the adhesive on oxygen diffusion that could slow the respiration rate (Fig. S1C). Calculations based on these measurements showed that c. 40 times more leaf tissue is required for an accurate OCR measurement in the typical 1-ml Clark-type oxygen electrode than in the microtitre plate fluorescence assay. All further experiments were performed using the Leukosan®/agarose mixture.
As a result of the need to fix the leaf discs in the wells, the preparation of a full 96-well plate takes c. 45 min. To test whether the order in which the leaf discs are laid down influences the reading, we used different leaf developmental stages, including slow and fast respiring stages, from two plants. Leaves and cotyledons were selected and the two sets of discs were fixed in the wells with a c. 30-min time difference between the sets (Fig. S2). Similar differences in respiration rate between the leaf stages were recorded. To test the dynamic range of the Seahorse XF96 instrument, an experiment was performed using different amounts of leaf tissue. As the leaf discs must be fixed to the bottom of the well, the maximal size of the leaf disc is limited by the diameter of the well, and only one leaf disc can be used. In addition to the leaf disc size used for all the other experiments described here (0.7 mg FW), four additional sizes were employed (Fig. S3A). The graph shows that the OCR increases linearly with increasing tissue amount (R2=0.873). In separate experiments using over 230 large leaf discs (1.6 mg FW), individual leaf disc values up to 500 pmol O2 min−1 were measured, the distribution of rates closely resembling a normal distribution (Fig. S3B). As the leaf discs used here are small and have a significant cut surface area to total surface area, an experiment was performed to test for a possible wounding-induced oxygen consumption effect on the readings. The standard procedure to reduce this effect by dark incubation was performed (Azcon-Bieto et al., 1983a,b; Day et al., 1985). Leaf discs were excised from three individual plants and incubated for 30 min in the dark in respiration buffer, before the measurements were performed (Fig. S4). No significant difference could be detected. All further experiments presented were performed without the 30-min dark incubation before adhering discs to the wells.
Respiration rate across Arabidopsis leaf surfaces
The ability to measure respiration in small leaf discs allowed us to survey the respiration rate of different regions across single Arabidopsis leaves. Nine 2.5-mm-diameter leaf discs were excised from three independent mature leaves of 4-wk-old A. thaliana plants to assess the respiration rate of the lamina left (L), lamina right (R) and mid-rib (M) positions on the leaf blade (Fig. 1). The mean OCR of each leaf disc position was assessed by averaging the mean OCR from three different leaves. On a leaf area basis, mid-ribs (M1–3) constantly showed a higher mean OCR than laminar positions, left (L1–3) and right (R1–3; Fig. 1b). Comparison of the mean OCR values showed that there were significant differences between mid-ribs (M2 and M3) and both laminar left (L1–3) and right (R1–3; P ≤0.05) positions. On a weight/volume basis, mid-ribs and lamina discs varied significantly, with c. 1.4-fold higher average FW of mid-rib leaf discs. As a result, mid-ribs exhibited a lower mean OCR than laminar leaf discs on a weight basis (Fig. 1c). Statistically significant differences between laminar left (L1–3) and mid-rib (M1 and M2) disc positions (P ≤0.05) were apparent in the data. This indicates that, where a leaf disc is cut across the Arabidopsis leaf surface, this can influence the OCR measured. The data also showed the consistency of measurements along the leaf blade for laminar and vascular regions.
Respiration rates in Arabidopsis leaves of different sizes and ages
To gain further insight into the effect of leaf age and leaf size on leaf OCR, assays on leaves across the rosette of 4- and 6-wk-old plants were performed (Fig. 2). The growth of A. thaliana plants was observed from when the cotyledons first started to expand. The sequence of subsequent leaf development was systematically recorded and all leaves were tagged for the final analysis phase. The OCR from each leaf was measured simultaneously in the microtitre plate assays to avoid any differences associated with time of day or time from leaf harvest. The data showed that OCR increased gradually from mature to immature leaves (linear R2 = 0.81, polynomial R2 = 0.85 at 4 wk; and linear R2=0.63, polynomial R2=0.84 at 6 wk), although there were also some fluctuations spanning across leaf age. The median OCRs were 155 pmol O2 min−1 per disc and 233 pmol O2 min−1 per disc for 4- and 6-wk-old plants, respectively. Interestingly, the peak OCR in leaf 13, initially noted in 4-wk-old plants, was maintained at 6 wk. After this point in development, new leaves appear to retain the same higher rate of respiration as leaf 13.
Respiration rates of root tips and expanding regions
Root growth on plates is commonly measured as a phenotype of Arabidopsis mutants and in assays analysing chemical effectors and nutritional responses (Migliaccio & Piconese, 2001; Oliva & Dunand, 2007). However, the very small mass of Arabidopsis roots often precludes biochemical measurements at the single root level. The differential rate of respiration in the growing tip and in the previously expanded regions is of interest, as it is considered to be an important factor in determining the root growth rate (Hanbury & Atwell, 2005). The OCRs of single root tips and single 5-mm sections of expanded roots were found to be sufficient to make accurate measurements using micro-respiratory techniques (Fig. 3a). The data showed that OCR was three times higher in root tips than in expanded root regions (Fig. 3b). The treatment of plants with NaCl has been reported to stimulate or inhibit the respiration of roots depending on the species studied (Jacoby et al., 2011). Treatments for only 10 min with 100 mM or 200 mM NaCl led to no significant change in respiration rate in our assays. By contrast, 400 mM NaCl for 10 min halved the respiration rate of single root tips (Fig. 3b). However, a time course of the respiratory response showed that 200 mM NaCl lowered the respiration rate over the first hour of treatment, whereas 400 mM NaCl stopped the respiration rate in root tips in the same time frame (Fig. 3c). This shows that, for the lower salt concentrations, time-dependent effects can be monitored using this respiration assaying system.
Respiration rates of Arabidopsis seeds and the respiratory response during germination and hormone treatments
The kinetics of respiration in seeds during germination has been studied in a variety of species, but is difficult in Arabidopsis because of seed size. Using 1 mg of Arabidopsis seeds, we measured the initiation of respiration during the first 60 min post-imbibition, and recorded a four-fold rise in OCR (Fig. 4a). The respiration of seeds could be inhibited significantly by the simultaneous injection of the respiratory poison KCN into the microtitre plate assays. The addition of the alternative respiratory pathway inhibitor SHAM failed to further inhibit OCR. This could either be a result of the difficulty of this compound in entering seeds or a lack of a significant alternative pathway rate early in the seed germination process. Previous studies have shown that alternative oxidase is induced during the second 24 h post-imbibition in Arabidopsis seeds (Narsai et al., 2011). To confirm that the OCR rise observed during this first hour is the initiation of respiration, we performed a study of control seeds and two seed treatments, one treatment involving pre-imbibition for 100 min and the other a 100°C heat treatment for 1 h (Fig. 4b). Pre-imbibed seeds immediately attained an OCR similar to the maximal rate over the 120 min of the experiment. Control seed OCR rose to this value over the first 40 min. Heat-treated seeds did not respire during the 120-min period.
By extending the time period for each respiratory measurement from 5 to 60 min (as outlined in Materials and Methods), we were able to modify the OCR assay to allow the measurement of the OCR for single seeds throughout the first 48 h post-imbibition. These assays showed that there are several phases of OCR during this 48-h period, beginning with a steady rise over the first 24 h, followed by a slowing of the rate of acceleration of OCR, and a subsequent rise in rate between 30 and 40 h post-imbibition (Fig. 4c). The addition of the germination-stimulating hormone GA3 increased the respiration rate during this 48-h period, but without any clear change in respiration kinetics. To determine whether abscisic acid (ABA) had a contrasting impact, we repeated this 48-h study and compared control seeds with ABA-treated seeds. Respiration of ABA-treated seeds was similar to that of untreated seeds for the first 2–3 h; OCR then remained constant until 12 h post-imbibition, but finally declined over the remaining time in the assay (Fig. 4d). These ABA-treated seeds did not visibly germinate in the 96-well plates, whereas the seeds that were not treated germinated normally during the measurement.
Technical limitations and advances for OCR measurements of plant tissues
For decades, researchers have been using Clark-type oxygen electrodes or infra-red gas analysers to measure the respiration rate from Arabidopsis cells and tissues (Noren et al., 1999; Hunt, 2003; Williams et al., 2008; Tomaz et al., 2010; Yang et al., 2011). In order to overcome the impact of the baseline drift value (c. 0.2 nmol min−1 in a typical 1-ml Clark-type oxygen electrode) and the differential needed between reference and sample gas streams in infra-red gas analyser measurements (> 5 ppm CO2 for accurate respiratory measurements), a minimum of 20–50 mg of plant tissue is normally needed for a single assay to avoid spurious results (Hunt, 2003; Meyer et al., 2009; Tomaz et al., 2010). As Arabidopsis tissues are much smaller in size than many other species used in plant research, the pooling of samples from different biological replicates has usually been required for respiratory measurements. This is not ideal and has limited the accuracy of studies that have focused on specific tissues at certain developmental stages. Because of this, it is not surprising that many reports find little if any differences in whole-tissue OCR between genotypes and/or treatments of Arabidopsis plants.
Fluorescence-based dispersed measurement of OCR in multi-well plate format offers high-throughput respirometry with a greatly decreased sample size requirement for each assay. We have shown that c. 40-fold less leaf tissue (FW c. 1 mg) can be used in a similar time frame to other assays (< 60 min). Through an extension of the time of methods, even single seeds can be assayed for their OCR. This approach allows for high sensitivity in OCR detection, a greater number of respiratory data points and extremely low sample mass requirements, which will be especially useful for respiratory studies of scarce biological samples from plants.
A significant issue for the use of the microtitre plate OCR assays in the Seahorse XF96 is the need to secure material during the mixing and measurement phases. This is especially problematic for plant leaves as they are gas-filled structures, and so their buoyancy needs to be overcome for an extended period of time and during the addition and mixing phases of the assays. Two different methods were tested to immobilize leaf discs onto microtitre plate bases with differing success. Cell-Tak (BD Bioscience) is a formulation of multiple polyphenolic proteins extracted from the blue mussel Mytilus edulis (Silverman & Roberto, 2007). Researchers have been using this adhesive protein mixture to immobilize animal cells and tissues for microplate assays for a number of years (Choi et al., 2010; Zhang et al., 2011; Robinson et al., 2012). However, Cell-Tak is expensive and we found that it took c. 30 min to adhere, leading to dehydration of leaf tissues which is undesirable. Cell-Tak also had a significant failure rate across wells in securing leaf tissues (c. 20% failure, Fig. S1). A much lower cost and more rapid solution was the use of medical-grade skin-glue (Leukosan®), which is nontoxic, sets in c. 2 min and, when mixed with agarose, provided an excellent adhesive for leaf tissues to plastic surfaces (< 5% failure, Fig. S1). The agarose also provided aeration on the side of the leaf disc in contact with the plastic, as agarose has a gas-permeable macroporous structure with pore sizes of 100–300 nm (Plieva et al., 2009). Larger scale multiplex assays using most or all of the 96 positions on a plate could be adhered, covered with respiration buffer and ready for assay by the Seahorse XF96 in c. 45 min using the agarose plus skin-glue method. The respiration rate was not greatly influenced by the order in which the samples were loaded (Fig. S2), or by wounding effects (Fig. S4), and it could be conducted over a dynamic range of c. 20 to 500 pmol O2 min−1.
Direct comparison of the readings for leaf discs from the Clark-type oxygen electrode and the Seahorse XF96 revealed overall higher values from the micro-respiratory technology in our hands. The discrepancy can be explained by various factors. The Clark-type oxygen electrode is a closed system, whereas the Seahorse technology is based on a semi-closed measuring environment which requires a range of diffusion calculations to be undertaken (Gerencser et al., 2009). As this device was developed for mammalian cell lines, it is equipped with a heater to ensure an optimal temperature of 37°C. Cooling is not possible and the lowest possible temperature in room temperature conditions is reported by the device as c. 28°C. A higher temperature leads to an increased respiration rate and could also contribute to the differences noted. Based on our experiments, we recommend the use of this technology to detect relative changes within a single plate or different plates using the same method. Comparisons between plates using different methods (e.g. measurement time) and between fluorescence-based micro-respiratory and Clark-type electrode assays tend to yield differences in absolute rate which are difficult to account for precisely, but show similar relative differences between biological samples.
Variations in leaf respiration rate across development
The architecture of leaf structures is closely related to their function, and thus is an important determinant of the primary productivity of plants (Fosket, 1994). Our results revealed that the OCR of the mid-rib vascular region is different from that of the lamina of Arabidopsis leaves on both a leaf area and leaf weight basis. The key physiological and structural differences between the lamina and mid-rib have been well addressed in leaves (Sylvester et al., 1996; Nelson & Dengler, 1997). Most fundamentally, this has shown that the ratio of spongy mesophyll to palisade is greatest in the mid-rib portion of the leaf and steadily decreases towards the leaf margin. Comparative data analysis of mitochondrial density in Arabidopsis tissue has shown that there is approximately half the mitochondrial volume (μm3 μm−3 tissue) in spongy mesophyll tissue than in palisade tissue (Armstrong et al., 2006a). A relatively sparse distribution of mitochondrial number in a higher cell volume could explain the mid-rib to lamina differences in OCR observed here. Tschiersch et al. (2012) used fluorescence measurements of oxygen concentration to image leaves, and noted that the concentration in intercostal regions of the leaf blade declined faster than in veins, and concluded that oxygen distribution was aligned to the structure in the leaf. This could be interpreted to mean that OCRs were faster in intercostal areas of the leaf (similar to our lamina leaf discs) relative to the veins (similar to our mid-rib region leaf discs); therefore, findings from both leaf discs and leaf imaging are in agreement.
Our data were consistent with a general trend of an increase in respiration rate from mature to immature leaves, independent of leaf size. Regression analyses indicated a relatively strong correlation between the two sets of variables in the plants tested (R ≥0.60). These data suggest that leaf aging changes the respiration rate in Arabidopsis. Jeong et al. (2004) showed this in aspen leaves, where OCR decreased by > 50% from young leaves to mature leaves. In Arabidopsis, immature, partially expanded leaves have been reported to show significantly higher rates of respiration compared with mature fully expanded leaves (Armstrong et al., 2006b). Our data provide a high-definition dataset showing the timing and extent of this phenomenon across the rosette. The reason for this difference most probably resides in a combination of mitochondrial number in leaves and metabolic demands in different leaves. The respiratory process is thought to assimilate nearly half of the total carbon gained from the photosynthesis process (Mogensen, 1977; Lambers, 1985; Amthor, 1989) and its consequence losses are equally shared between growth and maintenance processes during developmental stages (Amthor, 1984; Lambers, 1985). Growth respiration provides energy for the synthesis of new tissue throughout the developmental process, whereas maintenance respiration generates energy to be used for the synthesis of essential substances for existing tissues and metabolites for the survival and adaptation of plants under various environmental conditions (Lambers, 1985; Amthor, 1989). Previous findings have shown that the cost of maintenance respiration is comparable with the cost of growth in herbaceous plants, such as Arabidopsis. Once plant tissues reach maturation, the growth rate and respiration slow, and energy obtained from respiration mainly goes towards maintenance and transport processes (Amthor, 1984).
Spatial variation in root respiration rate
In this study, we showed that the small root tips of Arabidopsis have a nearly three-fold higher OCR when compared with a section of expanded root (Fig. 3). This is consistent with the expected higher energy demand in root tips, required for elongation, than in the expanded region of roots, or could relate to smaller vacuoles in the root tips. In Arabidopsis, mitochondrial mutants in the Lon1 protease (Solheim et al., 2012), in the membrane chaperone prohibitin (Van Aken et al., 2007) and in complex I subunits (de Longevialle et al., 2007; Meyer et al., 2009) all have short roots. To our knowledge, there is no precise information on the rate of respiration required to maintain root growth in Arabidopsis. However, we have reported recently that succinate dehydrogenase assembly factor 2 (sdhaf2) is needed for the assembly and activity of mitochondrial complex II and for normal root elongation in Arabidopsis (Huang et al., 2013). Whole-root respiratory assays showed no difference between wild-type and sdhaf2, but micro-respiratory measurements of root tips showed low oxygen consumption in sdhaf2, suggesting that a metabolic deficit is responsible for the decreased growth of the root tip (Huang et al., 2013). Micro-respiratory techniques could allow the measurement of root respiration in a range of mutants to determine whether root tip respiration is a major controller of root growth rate in Arabidopsis.
Studies of the response of whole-root systems to NaCl treatments have shown stimulatory (Shone & Gale, 1983; Burchett et al., 1984; Cramer et al., 1995) and inhibitory (Hwang & Morris, 1994; Epron et al., 1999) effects and, in some cases, no consistent response in respiration rate (Blacquiere & Lambers, 1981; Malagoli et al., 2008). Here, we found a consistent inhibition of OCR by increasing NaCl concentration and increasing time of exposure. Mixed respiratory responses to NaCl treatments in the variety of plant species studied may indicate that OCR in distinct regions of roots responds differently to salt (Jacoby et al., 2011). Dissection of the respiratory response of root tissues is evidently required to better understand the impact of saline conditions on the root system. The future use of micro-respiratory measurements to calculate root respiration and its response to combinations of different substrates or chemicals will aid our understanding of the physiological importance of respiration in defining root growth.
Kinetics of seed respiration during germination
The Arabidopsis seed OCR shown here has two phases during the germination process. One phase is seen from the onset of imbibition until 10–20 h post-imbibition, and most probably represents the physical hydration process. This first phase is followed by a short lag and then another phase of increasing respiration rate starting 20–30 h post-imbibition. This two-step phenomenon and its timing are consistent with the phases of metabolic initiation and mitochondrial biogenesis reported from Arabidopsis seed transcript profiling over the first 48 h post-imbibition (Narsai et al., 2011). We found that OCR of Arabidopsis seed was inhibited by > 70% by the respiratory poison KCN. This suggests that most of the respiration flux occurs via the cytochrome pathway in Arabidopsis mitochondria. The low level of participation of the alternative pathway of respiration may be supported by the lack of effect of the alternative pathway inhibitor SHAM (Lambers, 1985). The predominance of the cytochrome pathway during germination has also been reported in pea seeds and maize embryos, suggesting that this could be a conserved feature of respiration in a range of plant seeds (Alscher-Herman et al., 1981; Ehrenshaft & Brambl, 1990; Logan et al., 2001).
The phytohormones ABA and GA3 elicit a series of signal transduction pathways and normally show an antagonistic interaction. ABA controls dormancy maintenance, with ABA synthesis increasing to arrest germination until conditions are favourable for germination (Lopez-Molina et al., 2001; Reyes & Chua, 2007). By contrast, the synthesis of gibberellins is linked to germination initiation (Weitbrecht et al., 2011). In our experiments, the treatment of Arabidopsis seeds with GA3 increased the respiration rate significantly in the latter stages of the germination process. ABA treatments did not show an increase in OCR during the early stages after imbibition associated with the physical imbibition phase. However, ABA treatment showed a dramatic reduction in the OCR associated with the rest of the germination process. The suppression of OCR might be one of the mechanisms to regulate the germination process during hormonally regulated checkpoints. The capacity of this 96-well microtitre plate system to measure OCR of single Arabidopsis seeds over days, and their response to phytohormones, would allow the survey of seed OCR in libraries of Arabidopsis seeds during the germination process. As seeds germinate and survive the assay, this is a physiological, but nondestructive, assay system. This would make the micro-respiratory technique a powerful tool to develop phenotype screens of mutant and ecotype populations to help define regulators of the kinetics of respiration initiation during germination.
The adaptation of commercial, 96-well microtitre plate systems that measure OCR of plant tissues provides new opportunities for respiratory research. The small volume limit in the measurements in these instruments actually facilitates the analysis of key Arabidopsis tissues, and other small tissue samples from any plant species, that have often been particularly challenging in the past. By showing the dynamics of measurements made on leaves, root tips and seeds, we hope to stimulate research using these new tools. The potential for multiplexed micro-respiratory assays of up to 96 samples simultaneously means that the assay of mutant populations, phenotypic screens and wider ecotype comparisons in Arabidopsis may be possible in the future. This could provide new ways of combining molecular and physiological studies of respiration in plants.
This research was funded by support from the Australian Research Council (ARC) Centre of Excellence in Plant Energy Biology (CE0561495) to A.H.M. Y.S.S. was funded by a Malaysian Agricultural Research and Development Institute PhD scholarship, E.S. was funded as an ARC Australian Postdoctoral Fellow (DP110104865) and A.H.M. was funded as an ARC Future Fellow (FT110100242). C.H. and X.J. were funded by Fondecyt (1100601), Millennium Nucleus in Plant Functional Genomics (Plo-062-f) and a Conicyt Fellowship (21100640) to C.H.