Multiplex microrespiratory measurements of Arabidopsis tissues

Methods

Multiplex micro-respiratory measurements of Arabidopsis tissues

Yun Shin Sew1,2, Elke Str€oher1,2, Cristi�an Holzmann1,3, Shaobai Huang1,2, Nicolas L. Taylor1,2, Xavier Jordana3

and A. Harvey Millar1,2

1ARC Centre of Excellence in Plant Energy Biology, The University of Western Australia, Bayliss Building M316, 35 Stirling Highway, Crawley, WA 6009, Australia; 2Centre for Comparative

Analysis of Biomolecular Networks (CABiN), The University of Western Australia, Bayliss Building M316, 35 Stirling Highway, Crawley, WA 6009, Australia; 3Millenium Nucleus in Plant

Functional Genomics, Departamento de Gen�etica Molecular y Microbiolog�ıa, Facultad de Ciencias Biol�ogicas, Pontificia Universid�ad Cat�olica de Chile, Casilla 114-D Santiago, Chile

Author for correspondence:A. Harvey MillarTel: +61 8 6488 7245

Email: [email protected]

Received: 28 March 2013Accepted: 29 May 2013

New Phytologist (2013) 200: 922–932doi: 10.1111/nph.12394

Key words: Arabidopsis, leaves,mitochondria, respiration, root tips, seeds.

Summary

� 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 consump-

tion 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 phytohor-

mones gibberellic acid (GA3) and abscisic acid (ABA) on respiration during seed germination.� These studies highlight the potential for multiplexed micro-respiratory assays to study oxy-

gen 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.

Introduction

Plant cells rely on mitochondrial respiration for ATP, carbonskeletons for amino acid assimilation and organic acid buildingblocks for biosynthetic pathways. Respiration is the principalcomponent in CO2 loss from cells and is a key factor in theassessment of the carbon balance of plants and in definingthe factors influencing the plant growth rate (Amthor, 1989).The assessment of the cellular respiration rate therefore providesan important insight into the metabolic activity and physiologicalstate of plant tissues (Lambers, 1985). The respiration rate can bemeasured noninvasively as gas exchange from the surface of tis-sues 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 ofO2 concentration by polarographic Clark-type oxygen electrodesin closed systems (Walker, 1990; Hunt, 2003). CO2 productionhas been measured using gas-phase infra-red gas analysers inclosed systems or in differential open system configurations(Hill & Powell, 1968; Hunt, 2003). Micro-electrodes based onpolarographic 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 insidesingle photosynthetic cells (Bai et al., 2011). However, theseminiaturized methods are highly technical, low throughput,require substantial specialization and often involve painstakingadaptation for use on specific tissues of the target plant species.

Arabidopsis has now become the key model for understandingthe molecular components of respiration in plants. Most of ourrecent advances in the understanding of the biogenesis of mito-chondria and the retrograde regulation of respiration by intracel-lular signalling processes has originated from studies in thisspecies (Millar et al., 2011). However, reports of the measure-ment of the respiration rate of Arabidopsis, and how it is alteredwhen mitochondrial functions are changed, have been limited asa result of two key constraints. First, the small size of manyArabidopsis tissues has limited the options for the use of manyconventional gas exchange systems to measure respiration rates(and micro-respirometry, such as that reported in Arabidopsis sil-iques (Porterfield et al., 1999), is a very specialized field). Second,the lack of high-throughput assay systems has limited the full use

922 New Phytologist (2013) 200: 922–932 � 2013 The Authors

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Methods

of the resources in Arabidopsis biology to assess respiratory phe-notypes through the access of a wide range of mutants, ecotypesand tissue types.

The development of analyte-selective fluorophores, whichmonitor the partial pressure of oxygen, coupled to fibre-opticcables to monitor their fluorescent properties, has opened up newopportunities in respiratory measurements. Fluorophore-basedmicro-oxygen sensors have been used to monitor oxygen levelsinside plant seeds (Borisjuk & Rolletschek, 2009; Ast et al., 2012)and in the root rhizosphere (Rudolph et al., 2012) to studyhypoxia. These measurements are of oxygen concentration, notrespiration rate, and so diffusion of gases for specific tissuesneeds to be calculated or standardized for respiration rates to bededuced by time series measurements of oxygen concentration(Rudolph et al., 2012). Coupling fluorophore-based micro-oxygen sensors to microtitre plate assays, for which standardizeddiffusion rates can be calculated, has allowed the high-throughputanalysis of the respiration rate in milligrams of tissue in microlitrevolumes (Ferrick et al., 2008; Gerencser et al., 2009). Suchsystems have been commercialized and are now being used tomeasure cellular respiration rates and cellular bioenergetics ofisolated mitochondria and cells from mammalian tissues (Beesonet al., 2010; Rogers et al., 2011; Zhang et al., 2011, 2012).However, to our knowledge, the adaptation and use of suchsystems for intact plant tissues has not been tested systematically.

Here, we present optimized methods to adapt the use ofcommercial microplate assays of oxygen consumption by analyte-selective fluorophores to measure the respiration rates of Arabidop-sis leaf, root and seed samples. We show that this approach allowshigh-throughput measurements of the respiration rate in leaflaminar and vascular regions of a single leaf, the respiration rate ofsingle root tips and even the respiration of single imbibed seeds.We illustrate that biological changes in respiration associated withleaf development, leaf age, root segments and hormone-dependentchanges in seed germination can be measured and compared.These developments, and the use of commercial systems andconsumable packs already optimized and available to researchers,open up opportunities for the in-depth analysis of respiratoryphenotypes and their relation to developmental processes in smalltissue 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 basedon the fluorimetric detection of O2 levels via fluorophores in acommercial sensor cartridge. Oxygen quenches the fluorescenceof a fluorescein complex, the fluorescence is detected by afibre-optic waveguide and converted into the basal oxygenconsumption rate (OCR). During the ‘measurement’ phase, theconcentrations are measured continuously until the rate of changeis linear, and then OCR is determined from the slope. The probeslift whilst in the ‘mixing’ and ‘waiting’ steps to allow the largermedium 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 bevisualized and analysed in both XF96 Analyzer software and anExcel-based data viewer. For the underlying calculations, thereader is referred to the literature on the development of thissystem (Ferrick et al., 2008; Gerencser et al., 2009). Respirationmeasurements were performed in an XF96 Extracellular FluxAnalyzer (Seahorse Bioscience, Billerica, MA, USA) to obtain theOCR of plant tissues. The 96-well sensor cartridge was hydratedin 200 ll per well of XF Calibrant Solution (Seahorse Bioscience)overnight at 37°C before the assay. Several hours before the mea-surement commenced, the heater of the instrument was turned offto obtain a stable internal measurement temperature in the machineat c. 28°C. Plates (and injection ports when indicated) were filledusing multichannel pipettes or en masse by a 96-well robotic liquidhandling 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 (ecotypeColumbia) were placed on wet filter paper and incubated at 4°Cfor 3 d. The imbibed seeds were transferred to individual potscontaining a 1 : 3 perlite : soil mix and covered with a transparentacrylic hood to maintain humidity. The seedlings were grown ina controlled environment growth chamber maintaining a short-day photoperiod (8 h : 16 h, light : dark), a photon flux of150 lmol photons m�2 s�1, a relative humidity of 75% and atemperature cycle of 22°C : 17°C, day : night temperatureregime. When the seedlings were established, the acrylic hoodwas removed and the plants were subsequently grown with regu-lar watering. At an age of 4–6 wk, as indicated, the plants wereused for the measurements.

Single leaf discs were immobilized in wells with either Cell-Tak(BD Bioscience, North Ryde, NSW, Australia) or a commercialskin adhesive Leukosan® (BSN Medical, Mount Waverley, B.C.,VIC, Australia) mixed with agarose. For Cell-Tak adhesion, 16 llof the Cell-Tak mixture, pH 7 (5% (v/v) Cell-Tak, 45 mMsodium bicarbonate, pH 8.0), was used to coat the bottom of eachwell of the microtitre plate. The absorption of Cell-Tak to thewell bottom was allowed for 20 min at room temperature, afterwhich the Cell-Tak mixture was discarded by aspiration beforerinsing with distilled water. Single 2.5-mm-diameter leaf discs,which had been freshly cut with a leaf punch, were then placed atthe centre of each well and gently pressed to the well bottom usinga cotton bud. An even contact between the leaf disc and theCell-Tak-coated layer on the bottom of the well was required foroptimal adhesion. Adhesion was allowed for 30 min before 200 llof respiration buffer (10 mM HEPES, 10 mM MES and 2 mMCaCl2, pH 7.2 (Atkin et al., 1993; Armstrong et al., 2006a)) wasadded to the wells. For the skin-glue adhesive and agarose mix-ture, a combination of 2.5% (v/v) Leukosan® adhesive in 0.25%(w/v) agarose was prepared and kept above 60°C to avoid solidifi-cation. For each well, 1 ll of the adhesive mixture was pipettedonto the centre of the well bottom. Then, 2.5-mm-diameter leafdiscs were positioned on top of the mixture before gentle pressurewith a cotton bud. As the adhesive mixture sets in c. 2 min,

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sequential handling of the samples is required if large numbers ofleaf discs are used. After 2 min, respiration buffer can be added ontop of the leaf discs to avoid dehydration. A full plate of 96 leafdiscs could be manually adhered in c. 45 min. Once leaf adhesionhad been achieved, wells were filled with 200 ll of leaf respirationbuffer and loaded into the plate reader after the calibration steps.The time events for both basal respiration measurement andinjection were mixing (3 min), waiting (4 min) and measurement(5 min). The method allowed for 10 cycles of mixing, waitingand measurement. The OCR of single leaf discs was recorded bySeahorse XF Acquisition and Analysis Software (Version 1.3;Seahorse Bioscience).

Root respiration measurements by XF96

Seeds of Arabidopsis (A. thaliana) ecotype (Columbia-0) weresown on half-strength Murashige and Skoog (MS) Gamborg B5plates containing 0.8% (w/v) agar, 1% (w/v) sucrose, 1.8 mMMES at pH 5.8 adjusted by KOH. The plates were placed at 4°Cin the dark for 2 d and then transferred to a growth room with aphotoperiod of 16 h : 8 h, light : dark at a light intensity of200 lmol m�2 s�1, relative humidity of 70% and temperaturecycle of 22°C : 17°C, day : night. The plates were set in a verticalposition. After 7 d of growth, c. 5 mm of the expanded section orelongating root tip were cut for respiration assay with eight repli-cates for each treatment. The 96-well sensor cartridge was hydratedin 200 ll per well XF Calibrant Solution (Seahorse Bioscience) asmentioned above. After calibration, the 96-well utility plate wasfilled with 100 ll of respiration buffer containing 0, 100, 200 or400 mMNaCl. In each well, a single root tip (tip; c. 5 mm) or rootexpanded section (EXP; c. 5 mm) was added to the bottom of thewell. The time events for both basal respiration measurement andinjection were mixing (2 min), waiting (3 min) and measurement(5 min). Seven cycles of mixing, waiting and measurement wereapplied for time course measurements. The OCR of the single roottip or expanded section was recorded by Seahorse XF Acquisitionand Analysis Software (Version 1.3; Seahorse Bioscience).

Seed respiration measurements by XF96

For multiple seed measurements, intact seeds (c. 1 mg) wereplaced in a 96-well plate and surface sterilized by soaking for7 min in 12.5% (w/v) NaClO and 0.1% (v/v) Tween20, fol-lowed by two washing steps with distilled H2O. After this, wellswere filled with 200 ll of seed respiration medium (5 mMKH2PO4, 10 mM TES, 10 mM NaCl, 2 mM MgSO4, pH 7.2)and loaded into the plate reader after the calibration steps usingthe Bravo liquid handling station (Agilent Technologies). Whereindicated, inhibitors were added to the medium with a final con-centration of 2 lM for KCN or 5 mM for salicylhydroxamic acid(SHAM). Oxygen concentrations before and after inhibitor injec-tion were determined by 11 cycles of mixing (3 min), waiting(4 min) and measurement (5 min). The OCR of seeds wasrecorded by Seahorse XF Acquisition and Analysis software(Version 1.3; Seahorse Bioscience), and each well was normalizedby 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 ll) into the centre of eachwell bottom. Seeds were sterilized by overnight incubation withchlorine gas (100 ml of 12% NaOCl and 3 ml of 37% HClO) ina closed vessel. Each single seed was placed with a sterile tooth-pick, making sure the adhesion of each seed was in the centre ofthe well. Then, the wells were filled with 200 ll of seed respira-tion medium and loaded into the plate reader after the calibrationsteps. Where indicated, hormones were added to the respirationmedium with a final concentration of 2.4 lM 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 wasrun for 48 cycles, achieving a total of 50 h of measurements. TheOCR of single seeds was recorded by Seahorse XF Acquisitionand Analysis Software (Version 1.3; Seahorse Bioscience).

Leaf respiration by Clark-type oxygen electrode

Plants were grown under the conditions described for XF96above. The OCR of leaf discs was measured using a liquid-phaseOxygraph system (Hansatech Instruments, Pentney, Norfolk,UK). Before the measurement, the electrode was calibrated at25°C by the addition of sodium dithionite to 1 ml of aeratedautoclaved water to completely deplete oxygen. Leaf discs total-ling 40–60 mg fresh weight (FW) of 7-mm-diameter leaf discswere immersed in leaf respiration buffer and incubated in thedark for 30 min. Leaf respiration was performed in a 2-ml vol-ume for at least 15 min at 25°C in a darkened electrode chamber.The amount of oxygen being consumed by the leaf discs wasrecorded using Oxygraph Plus v1.02 software (HansatechInstruments), and the OCR (nmol min�1 g�1 FW) wascalculated accordingly to the FW of the leaf discs.

Statistical analysis

The statistical software package IBM SPPS Statistics 19 (IBMAustralia, St Leonards, NSW, Australia) was used for data analy-sis where indicated. An analysis of variance, followed by multiplecomparison using post hoc tests and Tukey’s honestly significantdifference (HSD) mean separation test, was performed todetermine the statistical significance of differences of the meanvalues at P ≤ 0.05.

Results

Adhesion of leaf discs for respiratory measurements

Making OCR measurements in microtitre plates of the SeahorseXF96 requires that the tissues remain at the bottom of the welland do not move during the cycles of mixing and measurement.This requirement is not present when using oxygen electrode orinfra-red gas analysis techniques, and is much less of a problemwhen using mammalian tissues as they are not buoyant

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structures. To develop suitable adhesion techniques, we trialledtwo 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 adhesionmethods were found to immobilize leaf discs submerged in bufferfor several hours. However, the investigation of the effectivenessof the two adhesion mixtures during the course of the mixingassays showed that the Leukosan® adhesive treatment producedfar fewer leaf disc detachment events and a lower standard errorfor OCR (Supporting Information Fig. S1A,B). Analysis showedthat an OCR of 143� 11 pmol O2 min�1 (for a leaf disc ofc. 0.7 mg FW) could be consistently measured. Replicate leafdiscs from the same leaf gave more consistent results than leaf-to-leaf comparisons, suggesting some variability of OCR betweenleaves (Fig. S1A,B). To test the effect of the adhesive on OCR,we performed similar measurements using 7-mm-diameter leafdiscs in a Clark-type oxygen electrode (Oxygraph; HansatechInstruments). The mean OCR g�1 FW of leaf discs did notchange with increasing amount of leaf discs adhered together dur-ing the analysis, indicating no substantial effect of the adhesiveon oxygen diffusion that could slow the respiration rate(Fig. S1C). Calculations based on these measurements showedthat c. 40 times more leaf tissue is required for an accurate OCRmeasurement in the typical 1-ml Clark-type oxygen electrodethan in the microtitre plate fluorescence assay. All further experi-ments were performed using the Leukosan®/agarose mixture.

As a result of the need to fix the leaf discs in thewells, the prepara-tion of a full 96-well plate takes c. 45 min. To test whether the orderin which the leaf discs are laid down influences the reading, we useddifferent leaf developmental stages, including slow and fast respir-ing stages, from twoplants. Leaves and cotyledonswere selected andthe two sets of discs were fixed in thewells with a c. 30-min time dif-ference between the sets (Fig. S2). Similar differences in respirationratebetween the leaf stageswere recorded.To test thedynamic rangeof the Seahorse XF96 instrument, an experiment was performedusing different amounts of leaf tissue. As the leaf discs must be fixedto the bottom of the well, the maximal size of the leaf disc is limitedby 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 experimentsdescribed here (0.7 mg FW), four additional sizes were employed(Fig. S3A). The graph shows that the OCR increases linearly withincreasing tissue amount (R2 = 0.873). In separate experimentsusing over 230 large leaf discs (1.6 mg FW), individual leaf discvalues up to 500 pmol O2 min�1 were measured, the distributionof rates closely resembling a normal distribution (Fig. S3B). As theleaf discs used here are small andhave a significant cut surface area tototal surface area, an experimentwas performed to test for a possiblewounding-induced oxygen consumption effect on the readings.The standard procedure to reduce this effect by dark incubationwasperformed (Azcon-Bieto et al., 1983a,b; Day et al., 1985). Leafdiscs were excised from three individual plants and incubated for30 min in the dark in respiration buffer, before the measurementswere performed (Fig. S4). No significant difference could bedetected. All further experiments presented were performed with-out the30-mindark incubationbefore adheringdiscs to thewells.

Respiration rate across Arabidopsis leaf surfaces

The ability to measure respiration in small leaf discs allowed usto survey the respiration rate of different regions across singleArabidopsis leaves. Nine 2.5-mm-diameter leaf discs were excisedfrom three independent mature leaves of 4-wk-old A. thalianaplants to assess the respiration rate of the lamina left (L), laminaright (R) and mid-rib (M) positions on the leaf blade (Fig. 1).The mean OCR of each leaf disc position was assessed by averag-ing the mean OCR from three different leaves. On a leaf areabasis, mid-ribs (M1–3) constantly showed a higher mean OCRthan laminar positions, left (L1–3) and right (R1–3; Fig. 1b).Comparison of the mean OCR values showed that there were sig-nificant differences between mid-ribs (M2 and M3) and bothlaminar left (L1–3) and right (R1–3; P ≤ 0.05) positions. On aweight/volume basis, mid-ribs and lamina discs varied signifi-cantly, with c. 1.4-fold higher average FW of mid-rib leaf discs.As a result, mid-ribs exhibited a lower mean OCR than laminarleaf discs on a weight basis (Fig. 1c). Statistically significantdifferences between laminar left (L1–3) and mid-rib (M1 and

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Fig. 1 Survey of Arabidopsis leaf bladerespiration rate excised from individualmature leaves of 4-wk-old Arabidopsis

thaliana plants. (a) The disc positions testedare depicted in the vertical (1, 2 and 3) andhorizontal (L, left; M, mid-ribs; R, right) axes.(b) Respiration rates on a leaf area basis.(c) Respiration rates on a leaf weight basis.The values represent the mean oxygenconsumption rate (OCR; n = 3; mean� SE).*, Significant difference (P ≤ 0.05) betweenM1–3 and the L1–3 and R1–3 bars.





M2) disc positions (P ≤ 0.05) were apparent in the data. Thisindicates that, where a leaf disc is cut across the Arabidopsis leafsurface, this can influence the OCR measured. The data alsoshowed the consistency of measurements along the leaf blade forlaminar and vascular regions.

Respiration rates in Arabidopsis leaves of different sizesand ages

To gain further insight into the effect of leaf age and leaf size onleaf OCR, assays on leaves across the rosette of 4- and 6-wk-oldplants were performed (Fig. 2). The growth of A. thaliana plantswas observed from when the cotyledons first started to expand.The sequence of subsequent leaf development was systematicallyrecorded and all leaves were tagged for the final analysis phase.The OCR from each leaf was measured simultaneously in themicrotitre plate assays to avoid any differences associated withtime of day or time from leaf harvest. The data showed thatOCR 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 somefluctuations spanning across leaf age. The median OCRs were155 pmol O2 min�1 per disc and 233 pmol O2 min�1 per discfor 4- and 6-wk-old plants, respectively. Interestingly, the peakOCR in leaf 13, initially noted in 4-wk-old plants, was main-tained at 6 wk. After this point in development, new leavesappear 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 ofArabidopsis mutants and in assays analysing chemical effectorsand nutritional responses (Migliaccio & Piconese, 2001; Oliva &Dunand, 2007). However, the very small mass of Arabidopsisroots often precludes biochemical measurements at the singleroot level. The differential rate of respiration in the growing tipand in the previously expanded regions is of interest, as it is con-sidered to be an important factor in determining the root growth

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(b)Fig. 2 The effects of development, leaf ageand leaf size on the oxygen consumption rate(OCR) of Arabidopsis thaliana leaves: (a) 4-wk-old plant and (b) 6-wk-old plant grownunder short-day conditions. The valuesrepresent the mean OCR (n = 4; mean� SE).The yellow lines indicate the calculatedmedian OCR and a colour scale was createdon the basis of the median for each plantage. The plant rosette and the size of eachleaf are shown in the images marked withleaf numbers. A colour scale assigned on thebasis of the calculated median aids thevisualization and comparison between thesize, developmental stage and rosetteposition of each leaf and its OCR value.Linear and polynomial lines of best fit areshown and R2 values are reported (linear,blue; polynomial, red).



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rate (Hanbury & Atwell, 2005). The OCRs of single root tipsand single 5-mm sections of expanded roots were found to be suf-ficient to make accurate measurements using micro-respiratorytechniques (Fig. 3a). The data showed that OCR was three timeshigher in root tips than in expanded root regions (Fig. 3b). Thetreatment of plants with NaCl has been reported to stimulate orinhibit the respiration of roots depending on the species studied(Jacoby et al., 2011). Treatments for only 10 min with 100 mMor 200 mM NaCl led to no significant change in respiration ratein our assays. By contrast, 400 mM NaCl for 10 min halved therespiration rate of single root tips (Fig. 3b). However, a timecourse of the respiratory response showed that 200 mM NaCllowered the respiration rate over the first hour of treatment,whereas 400 mM NaCl stopped the respiration rate in root tipsin the same time frame (Fig. 3c). This shows that, for the lowersalt concentrations, time-dependent effects can be monitoredusing this respiration assaying system.

Respiration rates of Arabidopsis seeds and the respiratoryresponse during germination and hormone treatments

The kinetics of respiration in seeds during germination has beenstudied in a variety of species, but is difficult in Arabidopsisbecause of seed size. Using 1 mg of Arabidopsis seeds, we

measured the initiation of respiration during the first 60 minpost-imbibition, and recorded a four-fold rise in OCR (Fig. 4a).The respiration of seeds could be inhibited significantly by thesimultaneous injection of the respiratory poison KCN into themicrotitre plate assays. The addition of the alternative respiratorypathway inhibitor SHAM failed to further inhibit OCR. Thiscould either be a result of the difficulty of this compound inentering seeds or a lack of a significant alternative pathway rateearly in the seed germination process. Previous studies haveshown that alternative oxidase is induced during the second 24 hpost-imbibition in Arabidopsis seeds (Narsai et al., 2011). Toconfirm that the OCR rise observed during this first hour is theinitiation of respiration, we performed a study of control seedsand two seed treatments, one treatment involving pre-imbibitionfor 100 min and the other a 100°C heat treatment for 1 h(Fig. 4b). Pre-imbibed seeds immediately attained an OCRsimilar 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 measurementfrom 5 to 60 min (as outlined in Materials andMethods), we wereable to modify the OCR assay to allow the measurement of theOCR for single seeds throughout the first 48 h post-imbibition.These assays showed that there are several phases of OCR duringthis 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 asubsequent rise in rate between 30 and 40 h post-imbibition(Fig. 4c). The addition of the germination-stimulating hormoneGA3 increased the respiration rate during this 48-h period, butwithout any clear change in respiration kinetics. To determinewhether abscisic acid (ABA) had a contrasting impact, we repeatedthis 48-h study and compared control seeds with ABA-treatedseeds. Respiration of ABA-treated seeds was similar to that ofuntreated seeds for the first 2–3 h; OCR then remained constantuntil 12 h post-imbibition, but finally declined over the remain-ing time in the assay (Fig. 4d). These ABA-treated seeds did notvisibly germinate in the 96-well plates, whereas the seeds that werenot treated germinated normally during the measurement.

Discussion

Technical limitations and advances for OCR measurementsof plant tissues

For decades, researchers have been using Clark-type oxygen elec-trodes or infra-red gas analysers to measure the respiration ratefrom 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 elec-trode) and the differential needed between reference and samplegas streams in infra-red gas analyser measurements (> 5 ppmCO2 for accurate respiratory measurements), a minimum of20–50 mg of plant tissue is normally needed for a single assay toavoid spurious results (Hunt, 2003; Meyer et al., 2009; Tomazet al., 2010). As Arabidopsis tissues are much smaller in size than

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Fig. 3 Respiration rates of single expanded region (EXP) and tip (TIP) of a7-d-old root of Arabidopsis thaliana seedlings. Plants were grown on agarplates under long-day conditions. (a) Single c. 5-mm sections of the rootexpanded region and root tip were used for each respiration assay. (b)Respiration rates of single root expanded region and root tip with orwithout different NaCl treatments for 10min (n = 5–8; mean� SE). (c)Time course of respiration rates of root tips treated with different NaClconcentrations (n = 5–8; mean� SE).





many other species used in plant research, the pooling of samplesfrom different biological replicates has usually been required forrespiratory measurements. This is not ideal and has limited theaccuracy of studies that have focused on specific tissues at certaindevelopmental stages. Because of this, it is not surprising thatmany reports find little if any differences in whole-tissue OCRbetween genotypes and/or treatments of Arabidopsis plants.

Fluorescence-based dispersed measurement of OCR in multi-well plate format offers high-throughput respirometry with agreatly decreased sample size requirement for each assay. We haveshown that c. 40-fold less leaf tissue (FW c. 1 mg) can be used ina similar time frame to other assays (< 60 min). Through anextension of the time of methods, even single seeds can be assayedfor their OCR. This approach allows for high sensitivity in OCRdetection, a greater number of respiratory data points andextremely low sample mass requirements, which will be especiallyuseful for respiratory studies of scarce biological samples fromplants.

A significant issue for the use of the microtitre plate OCRassays in the Seahorse XF96 is the need to secure material duringthe mixing and measurement phases. This is especially problem-atic for plant leaves as they are gas-filled structures, and so theirbuoyancy needs to be overcome for an extended period of timeand during the addition and mixing phases of the assays. Twodifferent methods were tested to immobilize leaf discs ontomicrotitre plate bases with differing success. Cell-Tak (BDBioscience) is a formulation of multiple polyphenolic proteinsextracted from the blue mussel Mytilus edulis (Silverman &Roberto, 2007). Researchers have been using this adhesive pro-tein mixture to immobilize animal cells and tissues for microplateassays for a number of years (Choi et al., 2010; Zhang et al.,2011; Robinson et al., 2012). However, Cell-Tak is expensiveand we found that it took c. 30 min to adhere, leading to dehy-dration of leaf tissues which is undesirable. Cell-Tak also had asignificant failure rate across wells in securing leaf tissues (c. 20%failure, Fig. S1). A much lower cost and more rapid solution wasthe use of medical-grade skin-glue (Leukosan�), which is non-toxic, sets in c. 2 min and, when mixed with agarose, provided anexcellent adhesive for leaf tissues to plastic surfaces (< 5% failure,Fig. S1). The agarose also provided aeration on the side of the leafdisc in contact with the plastic, as agarose has a gas-permeablemacroporous structure with pore sizes of 100–300 nm (Plievaet al., 2009). Larger scale multiplex assays using most or all of the

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Fig. 4 Respiration rates of Arabidopsis thaliana seeds. (a) Respiration rateof the first 110min post-imbibition of 1 mg of Col-0 seeds. Vertical linesindicate the time of addition of KCN (2 lM) and salicylhydroxamic acid(SHAM) (5 mM; n = 8; mean� SE). (b) Respiration rate of 1 mg of Col-0seeds which were untreated and assayed directly on imbibition (control),incubated in buffer at room temperature for 100min beforemeasurements (pre-treated) or heated at 100°C for 1 h in a buffer solutionbefore measurement (heat-treated; n = 8; mean� SE). (c) Respiration rateof single untreated Arabidopsis seeds and seeds incubated in 1.2mMgibberellic acid (GA3). Each seed was fixed to the centre of the well with0.25% (w/v) agarose (n = 14, mean� SE). (d) Respiration rate of singleuntreated Arabidopsis seeds and seeds incubated in 4 lM abscisic acid(ABA) Each seed was fixed to the centre of the well with 0.25% (w/v)agarose (n = 20, mean� SE).



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96 positions on a plate could be adhered, covered with respira-tion buffer and ready for assay by the Seahorse XF96 inc. 45 min using the agarose plus skin-glue method. The respira-tion rate was not greatly influenced by the order in which thesamples were loaded (Fig. S2), or by wounding effects (Fig. S4),and it could be conducted over a dynamic range of c. 20 to500 pmol O2 min�1.

Direct comparison of the readings for leaf discs from theClark-type oxygen electrode and the Seahorse XF96 revealedoverall higher values from the micro-respiratory technology inour hands. The discrepancy can be explained by various factors.The Clark-type oxygen electrode is a closed system, whereas theSeahorse technology is based on a semi-closed measuringenvironment which requires a range of diffusion calculations tobe undertaken (Gerencser et al., 2009). As this device was devel-oped for mammalian cell lines, it is equipped with a heater toensure an optimal temperature of 37°C. Cooling is not possibleand the lowest possible temperature in room temperature condi-tions is reported by the device as c. 28°C. A higher temperatureleads to an increased respiration rate and could also contribute tothe differences noted. Based on our experiments, we recommendthe use of this technology to detect relative changes within asingle plate or different plates using the same method. Compari-sons between plates using different methods (e.g. measurementtime) and between fluorescence-based micro-respiratory andClark-type electrode assays tend to yield differences in absoluterate which are difficult to account for precisely, but show similarrelative differences between biological samples.

Variations in leaf respiration rate across development

The architecture of leaf structures is closely related to their func-tion, and thus is an important determinant of the primary produc-tivity of plants (Fosket, 1994). Our results revealed that the OCRof the mid-rib vascular region is different from that of the laminaof Arabidopsis leaves on both a leaf area and leaf weight basis. Thekey physiological and structural differences between the laminaand mid-rib have been well addressed in leaves (Sylvester et al.,1996; Nelson & Dengler, 1997). Most fundamentally, this hasshown that the ratio of spongy mesophyll to palisade is greatest inthe mid-rib portion of the leaf and steadily decreases towards theleaf margin. Comparative data analysis of mitochondrial densityin Arabidopsis tissue has shown that there is approximately halfthe mitochondrial volume (lm3 lm�3 tissue) in spongy meso-phyll tissue than in palisade tissue (Armstrong et al., 2006a). Arelatively sparse distribution of mitochondrial number in a highercell volume could explain the mid-rib to lamina differences inOCR observed here. Tschiersch et al. (2012) used fluorescencemeasurements of oxygen concentration to image leaves, and notedthat the concentration in intercostal regions of the leaf bladedeclined faster than in veins, and concluded that oxygen distribu-tion was aligned to the structure in the leaf. This could beinterpreted to mean that OCRs were faster in intercostal areas ofthe leaf (similar to our lamina leaf discs) relative to the veins(similar to our mid-rib region leaf discs); therefore, findings fromboth leaf discs and leaf imaging are in agreement.

Our data were consistent with a general trend of an increase inrespiration rate from mature to immature leaves, independent ofleaf size. Regression analyses indicated a relatively strong correla-tion between the two sets of variables in the plants tested(R ≥ 0.60). These data suggest that leaf aging changes the respira-tion rate in Arabidopsis. Jeong et al. (2004) showed this in aspenleaves, where OCR decreased by > 50% from young leaves tomature leaves. In Arabidopsis, immature, partially expandedleaves have been reported to show significantly higher rates of res-piration compared with mature fully expanded leaves (Armstronget al., 2006b). Our data provide a high-definition dataset showingthe timing and extent of this phenomenon across the rosette. Thereason for this difference most probably resides in a combinationof mitochondrial number in leaves and metabolic demands indifferent leaves. The respiratory process is thought to assimilatenearly half of the total carbon gained from the photosynthesisprocess (Mogensen, 1977; Lambers, 1985; Amthor, 1989) andits consequence losses are equally shared between growth andmaintenance processes during developmental stages (Amthor,1984; Lambers, 1985). Growth respiration provides energy forthe synthesis of new tissue throughout the developmental process,whereas maintenance respiration generates energy to be used forthe synthesis of essential substances for existing tissues andmetabolites for the survival and adaptation of plants undervarious environmental conditions (Lambers, 1985; Amthor,1989). Previous findings have shown that the cost of maintenancerespiration is comparable with the cost of growth in herbaceousplants, such as Arabidopsis. Once plant tissues reach maturation,the growth rate and respiration slow, and energy obtained fromrespiration mainly goes towards maintenance and transportprocesses (Amthor, 1984).

Spatial variation in root respiration rate

In this study, we showed that the small root tips of Arabid-opsis have a nearly three-fold higher OCR when comparedwith a section of expanded root (Fig. 3). This is consistentwith the expected higher energy demand in root tips, requiredfor elongation, than in the expanded region of roots, or couldrelate 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 Akenet al., 2007) and in complex I subunits (de Longevialle et al.,2007; Meyer et al., 2009) all have short roots. To our knowl-edge, there is no precise information on the rate of respira-tion required to maintain root growth in Arabidopsis.However, we have reported recently that succinate dehydroge-nase assembly factor 2 (sdhaf2) is needed for the assemblyand activity of mitochondrial complex II and for normal rootelongation in Arabidopsis (Huang et al., 2013). Whole-rootrespiratory assays showed no difference between wild-type andsdhaf2, but micro-respiratory measurements of root tipsshowed low oxygen consumption in sdhaf2, suggesting that ametabolic deficit is responsible for the decreased growth ofthe root tip (Huang et al., 2013). Micro-respiratory techniquescould allow the measurement of root respiration in a range of





mutants to determine whether root tip respiration is a majorcontroller of root growth rate in Arabidopsis.

Studies of the response of whole-root systems to NaCl treat-ments have shown stimulatory (Shone & Gale, 1983; Burchettet al., 1984; Cramer et al., 1995) and inhibitory (Hwang &Morris, 1994; Epron et al., 1999) effects and, in some cases, noconsistent response in respiration rate (Blacquiere & Lambers,1981; Malagoli et al., 2008). Here, we found a consistent inhibi-tion of OCR by increasing NaCl concentration and increasingtime of exposure. Mixed respiratory responses to NaCl treat-ments in the variety of plant species studied may indicate thatOCR in distinct regions of roots responds differently to salt(Jacoby et al., 2011). Dissection of the respiratory response ofroot tissues is evidently required to better understand the impactof saline conditions on the root system. The future use of micro-respiratory measurements to calculate root respiration and itsresponse to combinations of different substrates or chemicals willaid our understanding of the physiological importance of respira-tion in defining root growth.

Kinetics of seed respiration during germination

The Arabidopsis seed OCR shown here has two phases during thegermination process. One phase is seen from the onset of imbibi-tion until 10–20 h post-imbibition, and most probably representsthe physical hydration process. This first phase is followed by ashort lag and then another phase of increasing respiration ratestarting 20–30 h post-imbibition. This two-step phenomenonand its timing are consistent with the phases of metabolic initia-tion and mitochondrial biogenesis reported from Arabidopsisseed transcript profiling over the first 48 h post-imbibition(Narsai et al., 2011). We found that OCR of Arabidopsis seedwas inhibited by > 70% by the respiratory poison KCN. This sug-gests that most of the respiration flux occurs via the cytochromepathway in Arabidopsis mitochondria. The low level of participa-tion of the alternative pathway of respiration may be supportedby the lack of effect of the alternative pathway inhibitor SHAM(Lambers, 1985). The predominance of the cytochrome pathwayduring germination has also been reported in pea seeds and maizeembryos, suggesting that this could be a conserved feature ofrespiration 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 signaltransduction pathways and normally show an antagonisticinteraction. ABA controls dormancy maintenance, with ABAsynthesis increasing to arrest germination until conditions arefavourable for germination (Lopez-Molina et al., 2001; Reyes &Chua, 2007). By contrast, the synthesis of gibberellins is linkedto germination initiation (Weitbrecht et al., 2011). In our experi-ments, the treatment of Arabidopsis seeds with GA3 increased therespiration rate significantly in the latter stages of the germinationprocess. ABA treatments did not show an increase in OCR dur-ing the early stages after imbibition associated with the physicalimbibition phase. However, ABA treatment showed a dramaticreduction in the OCR associated with the rest of the germinationprocess. The suppression of OCR might be one of the

mechanisms to regulate the germination process during hormon-ally regulated checkpoints. The capacity of this 96-well microtitreplate system to measure OCR of single Arabidopsis seeds overdays, and their response to phytohormones, would allow thesurvey of seed OCR in libraries of Arabidopsis seeds during thegermination process. As seeds germinate and survive the assay,this is a physiological, but nondestructive, assay system. Thiswould make the micro-respiratory technique a powerful tool todevelop phenotype screens of mutant and ecotype populations tohelp define regulators of the kinetics of respiration initiationduring germination.

Conclusions

The adaptation of commercial, 96-well microtitre plate systemsthat measure OCR of plant tissues provides new opportunitiesfor respiratory research. The small volume limit in the measure-ments in these instruments actually facilitates the analysis of keyArabidopsis tissues, and other small tissue samples from any plantspecies, that have often been particularly challenging in the past.By showing the dynamics of measurements made on leaves, roottips and seeds, we hope to stimulate research using these newtools. The potential for multiplexed micro-respiratory assays ofup to 96 samples simultaneously means that the assay of mutantpopulations, phenotypic screens and wider ecotype comparisonsin Arabidopsis may be possible in the future. This could providenew ways of combining molecular and physiological studies ofrespiration in plants.

Acknowledgements

This research was funded by support from the Australian ResearchCouncil (ARC) Centre of Excellence in Plant Energy Biology(CE0561495) to A.H.M. Y.S.S. was funded by a Malaysian Agri-cultural 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 FutureFellow (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.

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Supporting Information

Additional supporting information may be found in the onlineversion of this article.

Fig. S1 Comparison of leaf disc adhesion techniques on XF96cell culture microplates, and the effect of adhesives on Arabidopsisthaliana leaf respiration rate.

Fig. S2 Investigation of the effect on Arabidopsis thaliana leafrespiration rate of an alteration in the time at which discs wereadhered to the plates.

Fig. S3 Respiration rates of different sizes of leaf disc excisedfrom mature leaves of Arabidopsis thaliana plants.

Fig. S4 Investigation of the effect on Arabidopsis thaliana leafrespiration rate of the dark incubation of leaf discs to lowerwounding respiration.

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Multiplex microrespiratory measurements of Arabidopsis tissues