Methods
Methods
Multiplex micro-respiratory measurements of Arabidopsis tissues
Yun Shin Sew1,2, Elke Str€
oher1,2, Cristia n Holzmann1,3, Shaobai Huang1,2, Nicolas L. Taylor1,2, Xavier Jordana3
1,2
and A. Harvey Millar
1
ARC 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 Genetica Molecular y Microbiologıa, Facultad de Ciencias Biologicas, Pontificia Universidad Catolica de Chile, Casilla 114-D Santiago, Chile
Summary
Author for correspondence:
A. Harvey Millar
Tel: +61 8 6488 7245
Email: [email protected]
Received: 28 March 2013
Accepted: 29 May 2013
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doi: 10.1111/nph.12394
Key words: Arabidopsis, leaves,
mitochondria, respiration, root tips, seeds.
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.
Introduction
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 lowthroughput 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
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concentrations inside seeds and siliques (Porterfield et al., 1999)
and in root tissues (Armstrong et al., 2000). Recently microelectrodes 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
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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 microoxygen 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 analyteselective 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 microchamber, re-oxygenating the solution and thus restoring the
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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 ll 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 shortday photoperiod (8 h : 16 h, light : dark), a photon flux of
150 lmol 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 ll
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 ll
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 ll 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,
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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 ll 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 lmol 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 ll per well XF Calibrant Solution (Seahorse Bioscience) as
mentioned above. After calibration, the 96-well utility plate was
filled with 100 ll 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 ll 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 lM 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.
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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 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 ll 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 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 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.
Statistical analysis
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.
Results
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
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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-toleaf 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
L
M
pmol O2 min–1 disc–1
(a)
R
1
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*
*
*
M1
M2
M3
150
100
50
0
2
L1
L2
L3
R1
R2
R3
R1
R2
R3
Disc Position
3
5 mm
nmol O2 min–1 g–1
Fig. 1 Survey of Arabidopsis leaf blade
respiration rate excised from individual
mature leaves of 4-wk-old Arabidopsis
thaliana plants. (a) The disc positions tested
are depicted in the vertical (1, 2 and 3) and
horizontal (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 oxygen
consumption rate (OCR; n = 3; mean SE).
*, Significant difference (P ≤ 0.05) between
M1–3 and the L1–3 and R1–3 bars.
200 (b)
200 (c)
150
*
*
*
M1
M2
M3
100
50
0
L1
L2
L3
Disc Position
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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.
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 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
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
(a)
8
R² = 0.8453
pmol O2 min–1 disc–1
350
300
250
200
150
100
50
0
5
10
R² = 0.811
fast
13
3
15 16
14
15
1
8
5
6
7
2
11
16
12
10
4
3
13
11
7
9
4
6
slow
14
2
0
5
10
15
12
20
9
Leaf number (age decreasing)
0.5 cm
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
pmol O2 min–1 disc–1
(b)
350
300
250
200
150
100
50
0
R² = 0.6299 fast
R² = 0.8405
19
10
1112
1718
1314
21
20
22232425
16
26
27
15
3
0
9
4 5 6 7 8
5
slow
10
15
20
25
30
Leaf number (age decreasing)
0.5 cm
3 4
5
6
7
8
9
10
11
12
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13
14
15
16
17
18 19
20
21
22
23 24 25 26
27
Fig. 2 The effects of development, leaf age
and leaf size on the oxygen consumption rate
(OCR) of Arabidopsis thaliana leaves: (a) 4wk-old plant and (b) 6-wk-old plant grown
under short-day conditions. The values
represent the mean OCR (n = 4; mean SE).
The yellow lines indicate the calculated
median OCR and a colour scale was created
on the basis of the median for each plant
age. The plant rosette and the size of each
leaf are shown in the images marked with
leaf numbers. A colour scale assigned on the
basis of the calculated median aids the
visualization and comparison between the
size, developmental stage and rosette
position of each leaf and its OCR value.
Linear and polynomial lines of best fit are
shown and R2 values are reported (linear,
blue; polynomial, red).
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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
(a)
1 cm
EXP
(b)
pmol O2 min–1 per RS
TIP
120
90
60
30
pmol O2 min–1 per p
0
NaCl (mM)
(c)
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.
0
EXP
0
100
0
200
TIPS
100
400
200
400
120
90
60
30
0
10
20
30
40
50
60
70
Time (min)
Fig. 3 Respiration rates of single expanded region (EXP) and tip (TIP) of a
7-d-old root of Arabidopsis thaliana seedlings. Plants were grown on agar
plates under long-day conditions. (a) Single c. 5-mm sections of the root
expanded region and root tip were used for each respiration assay. (b)
Respiration rates of single root expanded region and root tip with or
without different NaCl treatments for 10 min (n = 5–8; mean SE). (c)
Time course of respiration rates of root tips treated with different NaCl
concentrations (n = 5–8; mean SE).
Ó 2013 The Authors
New Phytologist Ó 2013 New Phytologist Trust
Discussion
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
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928 Research
(a) 180
control
pmol min–1 mg–1
160
KCN
SHAM
140
120
100
80
60
40
20
0
0
20
40
60
80
100
120
Time (min)
(b) 300
pmol min–1 mg–1
250
200
pre-treated
150
heat-treated
control
100
50
0
0
20
40
60
80
100
120
140
Time (min)
(c) 120
pmol min–1 per seed
100
80
60
40
control
20
GA3
0
0
10
20
30
40
50
Time (h)
(d) 70
pmol min–1 per seed
60
50
40
control
30
ABA
20
10
0
0
10
20
30
Time (h)
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40
50
Fig. 4 Respiration rates of Arabidopsis thaliana seeds. (a) Respiration rate
of the first 110 min post-imbibition of 1 mg of Col-0 seeds. Vertical lines
indicate 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-0
seeds which were untreated and assayed directly on imbibition (control),
incubated in buffer at room temperature for 100 min before
measurements (pre-treated) or heated at 100°C for 1 h in a buffer solution
before measurement (heat-treated; n = 8; mean SE). (c) Respiration rate
of single untreated Arabidopsis seeds and seeds incubated in 1.2 mM
gibberellic acid (GA3). Each seed was fixed to the centre of the well with
0.25% (w/v) agarose (n = 14, mean SE). (d) Respiration rate of single
untreated 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).
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 multiwell 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 (Leukosanâ), 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
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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 (lm3 lm 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.
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Research 929
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
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930 Research
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 microrespiratory 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
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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.
Conclusions
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.
Acknowledgements
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.
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Supporting Information
Additional supporting information may be found in the online
version of this article.
Fig. S1 Comparison of leaf disc adhesion techniques on XF96
cell culture microplates, and the effect of adhesives on Arabidopsis
thaliana leaf respiration rate.
Fig. S2 Investigation of the effect on Arabidopsis thaliana leaf
respiration rate of an alteration in the time at which discs were
adhered to the plates.
Fig. S3 Respiration rates of different sizes of leaf disc excised
from mature leaves of Arabidopsis thaliana plants.
Fig. S4 Investigation of the effect on Arabidopsis thaliana leaf
respiration rate of the dark incubation of leaf discs to lower
wounding respiration.
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