CSIRO PUBLISHING

Functional Plant Biology, 2016, 43, 207219

http://dx.doi.org/10.1071/FP15253

Salt-stress induced alterations in the root lipidome of two barley

genotypes with contrasting responses to salinity
Siria H. A. Natera A,D, Camilla B. Hill B,C, Thusitha W. T. Rupasinghe A and Ute Roessner A,B
A

Metabolomics Australia, School of BioSciences, BioSciences 2, Parkville, Vic. 3010, Australia.

School of BioSciences, BioSciences 2, Parkville, Vic. 3010, Australia.
C
Present address: Western Barley Genetics Alliance, Western Australian State Agricultural Biotechnology Centre,
School of Veterinary and Life Sciences, Murdoch University, 90 South Street, Murdoch, WA 6150, Australia.
D
Corresponding author. Email: s.natera@unimelb.edu.au
B

Abstract. Changes in lipid metabolism and composition as well as in distinct lipid species have been linked with altered
plant growth, development and responses to environmental stresses including salinity. However, there is little information
available in the literature focusing on lipids in roots under soil-related stresses such as salinity. Barley (Hordeum vulgare L.)
is a major cereal grain and, as a glycophyte, suffers substantial yield loss when grown under saline conditions. Relatively
little is understood of adaptation and tolerance mechanisms involving lipids and lipid metabolism in barley roots during
development and under exposure to salinity stress. In this study we investigated the lipid composition of barley roots of
Clipper and Sahara two genotypes with contrasting responses to salinity before and after salinity stress using a
combination of three lipidomics techniques: Fatty acid compositional analysis, untargeted lipid proling, and targeted
analysis to prole quantitatively the individual molecular species of key plant lipid classes. Our results provide new insight
into the effect of salinity on fatty acid proles and key lipid classes within barley roots of two different genotypes, which
is discussed in the context of current knowledge of the root metabolic responses of cereal crops to salinity stress.
Additional keywords: Hordeum vulgare, lipid metabolism, lipidomics, mass spectrometry, salinity.
Received 21 August 2015, accepted 12 November 2015, published online 4 January 2016

Introduction
Plant lipids are structurally diverse hydrophobic or amphipatic
molecules that have a variety of biological functions including
in cell and membrane structure, transport, intracellular and
extracellular signalling, and carbon storage (Welti and Wang
2004; Wenk 2005). Lipids are generally classied into fatty
acids, glycerolipids, glycerophospholipids, sphingolipids, sterol
lipids, prenol lipids, saccharolipids and polyketides (Fahy et al.
2009). Fatty acid-derived molecules are important signalling
components in interspecies communication and in plant
defence, and are key precursor molecules for lipid, oxylipin
and plant hormone biosynthesis (Weber 2002). The bulk of
cellular membrane lipids are glycerophospholipids, which are
derived from phosphatidic acid (PA) and are composed of
different molecular species with varied fatty acid chain length
and degree of unsaturation (Narasimhan et al. 2013). The
major glycerophospholipids in plants are phosphatidylcholine
(PC), phosphatidylethanolamine (PE), phosphatidylglycerol
(PG), phosphatidylinositol (PI), phosphatidylserine (PS), and
lysophosphatidylcholine (LPC), which are important components
for the formation of lipid bilayers as part of cell membranes (van
Meer 2005). In addition to these lipids, sphingolipids, which are
derivatives of ceramides, as well as sterol and sterol esters, are
found in plant cell membranes.
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CSIRO 2016

Changes in lipid metabolism and composition, as well as of

distinct lipid species have been linked with altered plant
growth, development, and responses to environmental stresses
including salinity (Wang 2002; Meijer and Munnik 2003;
Mansour 2013; Okazaki and Saito 2014; Chalbi et al. 2015).
For example, evidence is accumulating that jasmonate and
derivatives, which originate from 18 : 3 (octadecanoid pathway)
and 16 : 3 (hexadecanoid pathway) fatty acids in the chloroplast,
contribute to salinity stress tolerance in plants (Weber 2002;
Walia et al. 2007; Yoon et al. 2009). Although lipidomics
analyses have been performed in plants (Devaiah et al. 2006;
Horn and Chapman 2011), few have specically targeted roots
(Chalbi et al. 2015). As roots are the main point of contact with
soil-related environmental and abiotic stresses such as salinity
and very little is known about root lipids in general and
specically in response to salt, this is an area of growing
research interest (Mittler 2006).
Barley (Hordeum vulgare L.) is a major cereal grain and
used as livestock feed, raw material for alcohol and starch
production, and food. With 130 million tons produced in 2012,
barley grain production ranks fourth behind rice, wheat and
maize (FAO 2012). Despite being a glycophyte and suffering
from considerable yield losses when exposed to saline growth
conditions, barley, as the most salinity tolerant cereal, is an
www.publish.csiro.au/journals/fpb

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Functional Plant Biology

established model plant for temperate cereals in salinity research

(Munns and Tester 2008). Previous analyses indicate that there
are major differences in metabolite type and abundance between
roots of cultivated plants and landraces and that they respond
differently to salinity on the physiological, molecular and
metabolic level (Widodo et al. 2009; Adem et al. 2014);
however, little is known about the effects of salinity on the
lipid composition of barley roots (Chalbi et al. 2015). Two
genotypes of barley, the malting cv. Clipper (Australia) and
the landrace (LR) Sahara (North Africa) are of particular
interest based on previously reported diversity in salt tolerance:
Shelden et al. (2013) found that in the early stages of seedling
germination (72 h post-germination), LR Sahara showed the
most signicant inhibition of root elongation but not of root
mass under 100 mM NaCl conditions, and cv. Clipper maintained
its relative root growth rate better. With longer term saltexposure, Widodo et al. (2009) found that when grown
hydroponically under controlled conditions, whereas both cv.
Clipper and LR Sahara showed similar initial reductions in
biomass after 3 weeks of 100 mM NaCl exposure, in the
following weeks, LR Sahara after 5 weeks of salinity treatment
was showing a recovery phenomenon and resumed growth and
cv. Clipper continued to show reduced growth relative to control
despite containing a sodium exclusion loci (Shi et al. 2010).
Because the study by Shelden et al. (2013) only investigated
early seedling stage growth responses under salinity, these
experiments were not of sufcient length to determine if LR
Sahara was capable of resuming growth after longer-term
exposure as seen in the study by Widodo et al. (2009). The
responses of barley to salinity stress were also shown to differ
between hydroponic and soil systems (Tavakkoli et al. 2010).
These observations indicate that developmental stage and growth
environment as well as the timing and length of exposure can
change the overall responses of plants to salinity stress.
Developments in high resolution, high sensitivity mass
spectrometry (MS) have facilitated the identication and
characterisation of key compounds in biological processes,
including metabolites, proteins and lipids (Lee et al. 2012;
Okazaki et al. 2013; Hill et al. 2014). Due to these recent
technological advances, almost all modern lipidomics approaches
utilise optimised and tailored MS-based methodologies (Horn
and Chapman 2012) and these have successfully been applied
to plant lipid research (Samarakoon et al. 2012; Shiva et al.
2013; Chalbi et al. 2015). Although these techniques on their
own provide rich biochemical and biological information,
comprehensive analyses that capture the complexity of lipid
composition in barley roots will help to develop a new level of
understanding of lipid metabolism within the context of
response to osmotic stresses including salinity stress.
Here, we report on a study of the lipidome extracted from
whole roots of Clipper and Sahara, two barley genotypes with
contrasting responses to salinity, to determine the range of
lipids present in barley roots and to investigate whether or not
they are altered following 5 weeks of exposure to salt. Plants
were grown hydroponically under control and salinity (100 mM
NaCl) conditions and were comprehensively analysed using
a combination of three lipidomics techniques: (i) fatty acid
compositional analysis using gas chromatography mass
spectrometry (GC-MS) was performed to quantify 24 fatty

S. H. A. Natera et al.

acids key to understanding lipid metabolism; (ii) untargeted

lipid proling using high performance liquid chromatography
coupled to quadrupole time-of-ight mass spectrometry (HPLCESI-QTOF-MS) was used to detect and semi-quantify 708
individual lipophilic mass features; and (iii) HPLC coupled to
triple quadrupole mass spectrometry (HPLC-ESI-QQQ-MS)
enabled the quantication of 64 known individual lipid species
from seven main plant lipid classes. Our results provide new
insights into the effects of salinity on fatty acid proles and
key lipid classes within barley roots of the two different
genotypes, which are discussed in the context of current
knowledge of the root metabolic responses of cereal crops to
salinity stress.
Materials and methods
Chemicals and lipid standards
All chemicals were purchased from Sigma-Aldrich (Castle Hill,
NSW, Australia) unless otherwise specied and all solvents
were purchased from Merck (Frenchs Forest, NSW, Australia)
and were of LC-grade or better unless otherwise specied. Lipid
standards (synthetic and puried) were from Avanti Polar Lipids
(Alabaster, AL, USA). Deionised water (18.2 MW) was produced
using a Synergy UV Millipore System (Millipore, Bayswater,
Vic., Australia) was used throughout.
Plant material
Genotypes of barley (Hordeum vulgare L.) were originally
sourced from the Australian Centre for Plant Functional
Genomics (Adelaide). Two genotypes of barley, the malting
variety Clipper (Australia) and the landrace Sahara 3771
(North Africa), were selected for lipidomics analysis based on
previously reported diversity in salt tolerance (Widodo et al.
2009; Shelden et al. 2013). These genotypes are parents of a
mapping population (Clipper  Sahara 3771, (Karakousis et al.
2003) and genetic resources are available for further genetic
characterisation.
Plant growth and treatment
Barley (Hordeum vulgare cv. Clipper and LR Sahara 3771)
seeds were surface sterilised with 70% ethanol for 1 min,
washed with deionised water (three times), incubated for
10 min in 1% hypochlorite solution and then rinsed at least
three times with deionised water. The seeds were then imbibed
in water under continuous aeration for 16 h at room temperature.
Germinated seeds were placed onto moist lter paper and kept
in the dark at 4C for 2 days before being returned to room
temperature and grown for 3 more days.
Individual seedlings were then transferred to plastic growth
containers lled with hydroponic solution and supported by
a piece of foam wrapped around the base of the stem. Between
24 and 26 plants were grown in each hydroponic container.
Plants were grown in controlled conditions with a 21C day/
18C night, a photoperiod regime of 16 h day/8 h night at
250 mmol m2 s1 photon ux intensity.
The nutrient solution (5 mM KNO3, 2 mM Ca(NO3)2, 2 mM
MgSO4, 0.5 mM Na2SiO3, 0.2 mM NH4NO3, 0.1 mM KH2PO4
and 0.05 mM NaFe(III) EDTA and the following micronutrients:
50 mM H3BO3, 10 mM ZnSO4, 5 mM MnCl2, 0.5 mM CuSO4 and

Lipidomics of barley roots in response to salt

0.1 mM NaMoO3) was aerated continuously and replaced

twice a week. Salt stress conditions were applied after 1 week
of growth in control conditions by incremental addition of
25 mM NaCl twice daily, up to the nal concentration of
100 mM NaCl along with additional CaCl2 to maintain ion
balance within the solution as previously described (Genc
et al. 2007). Roots were harvested 5 weeks after 100 mM
NaCl concentrations in the nutrient solution were reached,
patted dry quickly and then immediately frozen in liquid
nitrogen and stored at 80C until extraction.
Lipid extraction
Lipids were extracted from six replicates of control and salttreated Clipper and Sahara total root tissue using the following
chloroform/methanol extraction method: frozen roots were
roughly homogenised in liquid nitrogen using a mortar and
pestle. Frozen tissue powder (~60 mg, accurate weight was
recorded) was transferred into cryo-mill tubes (Retsch GmbH,
Haan, Germany) and homogenised for 3  45 s in a cryo-mill
(Precellys 24; Bertin Technologies, le-de-France, France).
After the addition of 150 mL methanol and 300 mL chloroform,
the samples were incubated for 15 min at 37C in a thermomixer
at 750 rpm. Following centrifugation for 10 min at 13 800g
at room temperature, the supernatant was transferred into a
reaction tube, dried in vacuo and stored under nitrogen at
80C until analysis by GC-MS, HPLC-ESI-QTOF-MS or
HPLC-ESI-QQQ-MS as described below. Pooled biological
quality control (PBQC) samples were prepared by pooling
equal aliquots of each individual sample, which were used for
method optimisation and quality control as described in Hill
et al. (2014).
Fatty acid analysis by GC-MS
Fatty acid composition of the root lipid fractions was determined
by GC-MS analysis following methanolysis and trimethylsilyl
(TMS) derivatisation essentially as per Olmstead et al. (2013)
with the following modications. Lipid extracts were resuspended
in 25 mL of chloroform : methanol (2 : 1 v/v) containing 60 mM of
internal standard (13C-labelled myristic acid) before the addition
of 5 mL of derivatising agent (Meth-Prep II Grace Davison
Discovery, Deereld, IL, US). The sample was incubated at
30C for 30 min followed by a 10 min hold at room temperature
before 1 mL was injected onto a GC-MS system consisting of a
Gerstel 2.5.2 autosampler, a 7890A Agilent gas chromatograph
and a 5975C Agilent quadrupole MS (Agilent Technologies,
Santa Clara, CA, USA). The GC was performed on a 30 m
column with a 0.25 mm lm thickness, 0.25 mm inner diameter
and a 10 m guard column (Agilent JandW Scientic VF-5MS
GC Column). The injection port temperature was set to 250C,
the MS transfer line at 280C, the ion source adjusted to 230C
and the quadrupole at 150C. Helium was used as the carrier
gas at a ow rate of 1.0 mL min1. For fatty acid methyl ester
(FAME) analysis, the temperature program was as follows; start
at injection 50C, a hold for 1 min, followed by a 15C min1 oven
temperature ramp to 230C, hold for 3 min followed by a 10C
min1 oven temperature ramp to 325C and a nal 3 min heating
at 325C. Chromatograms and mass spectra were evaluated
using MSD Chemstation (ver. E.02.02.1431) and MassHunter

Functional Plant Biology

209

MS Quantitative Analysis (ver. B.06.00), software packages

(Agilent Technologies). Mass spectra of eluting FAMES were
identied and compared with authentic fatty acid standards on
the basis of mass spectral and retention time matching. The
concentration of each FAME were determined for the selected
ion area compared with a calibration curve of its respective
standard and then normalised to FW of the starting tissue and
are expressed as picomoles per mg fresh weight. All the GC-MS
data are presented as a mean of six biological replicates for each
treatment group (n = 6)  s.e.
Untargeted lipid proling
Lipid proles of the individual root lipid extracts, prepared as
described in the lipid extraction section, were obtained by
analysis using HPLC-ESI-QTOF-MS. Dried lipid extracts
were re-suspended in butanol/methanol (1 : 1, v/v) containing
5 mM ammonium formate and 1 mL aliquots were injected onto
a 150 mm  2.1 mm  2.7 mm Poroshell 120 EC-C8 column
(Agilent Technologies) at 35C using an Agilent LC 1290
(Agilent Technologies). Lipids were eluted at 0.26 mL min1
over 30 min gradient of water/acetonitrile (40 : 60, v/v) to
acetonitrile/isopropanol (10 : 90, v/v) with a binary gradient as
described by Hu et al. (2008) and analysed by electrospray
ionisation-mass spectrometry (ESI-MS) using a QTOF 6550
(Agilent Technologies). The separated lipid species from the
column were detected in positive mode and fragmentation
voltage was set as 175 V, sheath gas temperature was set as
275C and sheath gas ow was maintained at 12 L min 1. The
capillary voltage and nozzle voltage were set as 4000V and
1000V respectively. The temperature of the nitrogen drying
gas was set at 250C and drying gas ow was maintained at
13 L min 1. The LC-MS data presented represent values of six
biological replicates for each treatment group.
After initial visual inspection and data integrity checks
using Agilent Mass Hunter Qualitative Analysis software (ver.
B.06.00), the LC-QTOF-MS data comprising of features
(m/z_RT, mass of ion_retention time) and associated peak
intensities was converted to mzData format for processing
using MZmine 2.11 (http://mzmine.sourceforge.net/, accessed
10 September 2014) (Pluskal et al. 2010) and statistical
analysis using MetaboAnalyst 3.0 (http://www.metaboanalyst.
ca/, accessed 22 April 2015) (Xia et al. 2015). mzData export
was done using default values for centroid data with the
exception of MS peak lter being set to absolute height and a
count threshold of 2000. This threshold was determined by
prior manual inspection of the data and was ~10 times noise
level in the chromatograms. Univariate analysis of the data was
done using one-way ANOVA and post-hoc analysis using
Tukeys honestly signicant difference (HSD) test. Additional
statistical analysis (Students t-tests) comparing each genotype
with and without salt-treatment was also performed using
MetaboAnalyst to generate a list of features (m/z_RT) that
showed a signicant (P < 0.01) fold change of two or more in
peak intensity relative to the respective controls. In addition, a
pooled biological replicate made up aliquots of all the samples
(Hill et al. 2014) was also run in a data-dependent or auto
MS/MS mode to facilitate the identication of mass features
containing fragments characteristic of specic lipid classes.

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Functional Plant Biology

S. H. A. Natera et al.

Possible lipid assignments were made for mass features of

interest using the Lipid Maps (Fahy et al. 2007b) online tool
(http://www.lipidmaps.org/tools/ms/lm_mass_form.php, accessed
15 May 2015). Peak lists were searched as positive mode data
using a  0.01 Da m/z mass error considering [M+H]+, [M+Na]+
and [M+ NH4]+ as possible adducts.

Table 1. Lipid standards used for targeted (QQQ) lipidomics analysis

For full IUPAC names see Table S1, available as Supplementary Material to
this paper. Abbreviations: LPC, lysophosphatidylcholine; PA, phosphatidic
acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG,
phosphatidylglycerol; PI, phosphatidylinositol; PS, phosphatidylserine; d,
deuterated

Targeted lipid analysis

Lipids species present in the root lipid extracts used for untargeted
lipid proling were further analysed and quantied by
HPLC-ESI-QQQ-MS/MS essentially as described by Zhou
et al. (2014). The extracted lipids were analysed using a 1200
series HPLC coupled to a 6410B ESI-QQQ-MS (Agilent
Technologies). An injection of 15 mL of each total lipid
extract was chromatographically separated on an Ascentis
Express RP-Amide 50  2.1 mm, 2.7 mm HPLC column (SigmaAldrich, Castle Hill, NSW) using an 8 min gradient from
0% A to 100% B, which was then held for 2 min and followed
by a 4 min column re-equilibration with a ow rate of
0.2 mL min 1. The mobile phases were: A, 10 mM ammonium
formate in water : methanol : tetrahydrofuran (50 : 20 : 30, v/v/v); B,
10 mM ammonium formate in water : methanol : tetrahydrofuran
(5 : 20 : 75, v/v/v).
The molecular species of the following lipid classes were
identied using precursor ion scanning from 100 to 1200 m/z
in positive ion mode; phosphatidylcholine (precursors of m/z
184.1), sphingomyelin (m/z 184.1), ceramide (m/z 264.6),
cholesterol esters (m/z 369.4), phosphatidylglycerol (m/z 189)
and in negative ion mode; phosphatidylinositol (m/z 241). Neutral
loss scanning was used to identify phosphatidylethanolamine
(in positive ion mode, neutral loss of m/z 141), phosphatidylserine
(negative ion mode, neutral loss of m/z 87).
Identied lipid species were quantied using multiple
reaction monitoring (MRM) with a 10 ms dwell time for the
simultaneous measurements of up to 100 compounds. We
used optimised parameters for capillary (4000 V), fragmentor
(60160 V) and collision voltages (2040 V). In all cases, the
collision gas was nitrogen with a ow rate of 7 L min 1.
MS data was processed using Agilent Mass Hunter Qualitative
Analysis software (ver. B.06.00) for scan data and Quantitative
Analysis software for QQQ (ver. B.06.00) for MRM data
(Agilent Technologies). Lipids were quantied using external
standards listed in Table 1 (Full IUPAC names listed in Table S1,
available as Supplementary Material to this paper) (Avanti Polar
Lipids) injected in the same batch and presented as mol% in
the total lipid extract. All LC-MS data are presented as mean
of replicate samples (n = 6) with standard error. Values were
calculated as picomoles mg1 FW and are presented as mol%.
Detected lipid species are annotated as follows: lipid class
designation (total number of carbon atoms in the fatty acid
chains: total number of double bonds in the fatty acid chains).

Avanti catalogue number

Results
Changes of fatty acid proles of barley roots upon salinity
stress
The fatty acid composition of the root lipid extracts from the
two barley genotypes Clipper and Sahara with contrasting
responses to exposure to salt (100 mM NaCl for 5 weeks) was

855676
830856P
860399
830756
860384
840044P
840028P

Lipid
LPC (17 : 0)
PA (34 : 0)
PC (34 : 1)d
PE (34 : 0)
PG (16:0/18:1)d
PI (34 : 2)
PS (34 : 0)

determined by GC-MS of the released fatty acid methyl esters.

Fig. 1 shows absolute concentrations of 24 fatty acids that were
measured with identities conrmed via comparison to authentic
standards (Fig. 1; Table S2). Levels of other fatty acids that
were detected but could not be identied via comparison to
authentic standards will not be described here.
The predominant fatty acid species identied in barley roots
were C16:0, C18:2, C18:3 and C18:0, present at concentrations
between 830 and 1680 picomoles mg1 FW in the barley roots
(see Table S3). Despite being present at much lower abundance
(between 3.8 and 30 picomoles mg1 FW), the root lipid extracts
were also found to contain odd (C15, C17, C21 and C23) and
long carbon chain fatty acids with a chain length up to C24.
Salinity stress had only a small impact on the quantity of fatty
acids in barley roots: In roots of the North African landrace
Sahara, the fatty acid levels of C16:1 (1.61-fold), C18:2n6c
(1.12-fold) and C23:0 (1.27-fold) decreased signicantly
after treatment with 100 mM NaCl, whereas C18:3n6 (1.32fold) and C20:3n6 (1.1-fold) increased. In roots of the
Australian malting cultivar Clipper, the fatty acid levels of
C15 (-1.70-fold) and C16:1 (-2.04-fold) decreased, whereas
C12:0 (1.09-fold), C20:0 (1.16-fold) and C22:6n3 (1.16-fold)
increased signicantly after treatment with 100 mM NaCl. The
only common fatty acid change was C16:1, which decreased
in both genotypes (2-fold in Clipper and 1.7-fold in Sahara)
following salinity treatment when compared with their
respective controls.
Clipper roots show more lipid prole changes following
exposure to salt than Sahara
To study the effect of salinity stress on the global root lipid
prole, lipids extracted from whole roots of cv. Clipper and LR
Sahara after exposure to 100 mM NaCl for 5 weeks and from
equivalently aged control plants grown in the same experiment
under normal salt conditions were compared by untargeted
lipid proling. This approach involved separation of complex
mixtures of lipids by high performance liquid chromatography
coupled to quadrupole time-of-ight mass spectrometry (HPLCESI-QTOF-MS) to analyse a wide range of lipid classes in a
single run. The obtained proles were then compared to
investigate differences between the root lipids of the different
barley genotypes with contrasting responses to salt and to

Lipidomics of barley roots in response to salt

Functional Plant Biology

C12:0

C14:0

C15:0

7.5

10.0

5.0

7.5

1500
30
1000

20

5.0
2.5

2.5

C16:1

10

500

C17:0

C18:0

C18:1n9c

6
40

100

20

C18:1n9t

C18:2n6c

C18:3n3

1500

C18:3n6

8
1000

20
1000
10
0

4
500

500

C20:0

C20:1

C20:2

C20:3n6

10.0

*
7.5

*
4

7.5

7.5

5.0

5.0

2.5

2.5

2.5

C22

C22:1n9

15

10

C22:6n3

2
0

C23:0

C24:0

15

20

10

15

C24:1
9

10

4
5

0
S
h
Sa

h
Sa

C
lip
C

C
Sa

h
Sa

lip
C

lip

S
h
Sa

h
Sa

lip
C

lip
C

0
C

C
lip

C22:2

Sa
h

20

C21:0

lip

5.0

10.0

200
4

C
lip

10

Sa

15

Picomoles mg1 FW

C16:0

12.5

Fig. 1. Concentrations of individual fatty acids in root lipid extracts from the barley genotypes Clipper and Sahara after 5 weeks of
salinity stress (100 mM NaCl) compared with their respective controls as measured by GC-MS. Abbreviations: Clip C, Clipper control;
Clip S, Clipper salt-treated; Sah C, Sahara control; Sah S, Sahara salt-treated. Values are means of six replicate samples for each treatment
group (n = 6)  s.e. Fatty acid designation indicates number of carbon atoms: number of double bonds in the fatty acid chain. Full fatty
acid IUPAC names are listed in Table S2 (available as Supplementary Material to this paper).

211

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Functional Plant Biology

S. H. A. Natera et al.

identify lipid classes and species that were altered to be potentially

targeted for further analysis.
Multivariate analysis of the resultant root lipid proles using
hierarchical cluster analysis (HCA) showed clear separation of
the Clipper and Sahara genotypes as well as separation between
the control and salt-treated proles from each genotype (see
Fig. S1, available as Supplementary Material to this paper).
ANOVA and post-hoc analysis of the 708 features (m/z_RT,
retention time) extracted from root lipid proles showed that
more than half (378 or ~53%) were signicantly altered (P < 0.05)
between the four treatment groups to various extents. Signicant
differences from the lipid prole comparisons are summarised
in Fig. 2.
Exposure to salt stress resulted in a greater number of
signicant changes in the lipid prole of whole cv. Clipper
(253 features) roots compared with LR Sahara (121 features)
roots. Of these signicantly altered features, 89 were found to
be changes common to both genotypes following salt treatment
(Fig. 2). Comparison of the lipid proles from Clipper and
Sahara roots grown under control conditions showed a larger
number of signicantly altered features (246 features) than
between the lipid proles of the salt-treated roots of the two
genotypes (217 features). This suggests that there may be a
larger number of putative lipids that are inherently different
between the genotypes than those differently altered in the two
genotypes following salt-treatment (genotype specic responses
to salt).
To gain great insight into the directionality of these changes
more stringent statistical analysis was carried out on the two
genotypes and their respective salt-treated samples in a pairwise
analysis with the summary of the changes shown in Table 2.
Analysis of mass features signicantly changed (P < 0.01,
according to Students t-test) by 2-fold or more indicated that
of the 708 features extracted from the lipid proles, 152 were
found to be signicantly changed (P < 0.01) by 2-fold or more

Clipper
Control

246

Sahara
Control

253

Clipper
Salt

89

217

121

Sahara
Salt

Fig. 2. Venn diagram summary of signicantly altered features identied

by ANOVA, post-hoc analysis of proles of root lipid extracts from the
barley genotypes Clipper and Sahara after 5 weeks of salinity stress (100 mM
NaCl) when compared with their respective controls as measured by lipid
proling using HPLC-ESI-QTOF-MS. Numbers appearing in overlapped
areas between circles are the number of features with signicant changes
between those two treatments, e.g. there were 217 signicant changes
between Clipper Salt and Sahara Salt. The number 89 in the middle
represents the number of signicant changes that additionally occur in the
comparison between Clipper Control with Clipper Salt, as well as Sahara
Control with Sahara Salt: n = 6 for each treatment group, e.g. Clipper Control.

in Clipper roots after salt treatment when compared with control

roots (see Tables S4S6). In keeping with the multivariate
analysis, cv. Clipper showed a larger proportion of signicant
changes after salt treatment than Sahara where 86, or nearly
57%, of these decreased whereas the remaining 66 (~43%) were
signicantly increased by 2-fold or more. We noted that in the
LR Sahara roots, this trend was reversed, with 36% of the 61
signicantly altered features (greater or equal 2-fold change,
P < 0.01) being decreased with the majority (64%) increased.
Cv. Clipper showed many more signicantly altered features
following salt (152 compared with 61 in LR Sahara). There were
41 features in common with fold-changes in the same direction
(i.e. increased in both genotypes following salt treatment).
Therefore, 111 were considered to be unique changes in cv.
Clipper and 20 features unique to the LR Sahara root response
after salt.
All the features deemed to be signicantly different between
the sample groups were searched against the LipidMaps database
(Fahy et al. 2007a) to determine putative lipid assignments
based on precursor mass (Table 3). The resulting putative lipid
Table 2. Untargeted lipid proling (HPLC-ESI-QTOF-MS) features
signicantly altered in lipid extracts of Clipper and Sahara barley roots
following salt treatment relative to controls
Students t-test, P < 0.01, fold change (FC) 2. n = 6 for each treatment
group. For details see Tables S4S6, available as Supplementary Material
to this paper
Features altered in
both genotypes
following salt
Increased
14

Clipper Control vs
Clipper Salt

Decreased
27

Increased
72

Decreased
39

Sahara Control vs
Sahara Salt
Increased
9

Decreased
11

Table 3. Putative LipidMaps (Fahy et al. 2007a) lipid class assignments

for features signicantly altered in Clipper and Sahara barley roots
after salt-treatment compared with their respective controls (pairwise
comparisons, P < 0.01, fold-change $2) as determined by comparison
of lipid proles obtained using HPLC-ESI-QTOF-MS as described in
Materials and methods
For each treatment group (e.g. Clipper Control): n = 6. Abbreviations: PL,
phospholipid; GL, glycerolipid; CL, cardiolipin; FA, fatty acid; LPL,
lysophospholipid; PIP, phosphoinositolphosphate; CerP, ceramide
phosphate. For details see Table S7, available as Supplementary Material
to this paper
Lipid class

Common

Uniquely
altered in
Clipper
Count
%

Uniquely
altered in
Sahara
Count
%

Count

PL
GL
CL
FA
LPL
PIP
CerP

24
5
3
2
1
1
0

67
14
8
6
3
3
0

44
11
3
3
1
1
1

69
17
5
5
2
2
2

7
1
1
0
2
1
0

58
8
8
0
17
8
0

Total

36

100

64

100

12

100

Lipidomics of barley roots in response to salt

assignments were then broadly grouped into different lipid

classes including phospholipids such as PCs, PEs, PGs,
glycerolipids such as diacylglyerols (DAGs) and triacylglycerols
(TAGs), cardiolipins, lysophospholipids and ceramide phosphates.
Despite the larger number of signicant differences
apparently unique in cv. Clipper following salt treatment, of
the mass features with putative lipid assignments, a very
similar proportion (67% or 44 of the 64 features that returned
possible matches) were assigned as possible phospholipids with
a similar trend in LR Sahara (58%, or 7 out of 12 features that
returned possible matches were classied as phospholipids) as
shown in Table 3.
Of the 41 features signicantly altered following salt in both
genotypes, 36 returned LipidMaps putative lipid assignments
based on matches within 0.01 Da, considering protonated
species ([M+H]+) only. Of these, most were tentatively
assigned as phospholipids (24 out of 36) although the specic
phospholipid class could not be assigned due to the isobaric
nature of many phospholipids. This indicated that further,
targeted lipid analysis was required to determine specic
phospholipid class and species assignment.
Salinity stress results in signicant changes in many
phospholipid species
To improve lipid class and species assignments and
quantication to aid interpretation of the data, targeted lipid
analysis using liquid chromatography combined with triple
quadrupole mass spectrometry (HPLC-ESI-QQQ-MS/MS)
was used to further analyse the total lipid extracts from
control and salt-stressed roots of the two barley genotypes
Clipper and Sahara. Sixty-three individual phospholipid
species were quantied using this approach. Fig. 3 shows
phospholipids quantied in Clipper and Sahara roots
following salt treatment compared with their respective
controls as mole percent (mol%) values.
The phosphatidylcholines (PCs) were the most abundant
class of phospholipids measured in barley roots (~440690
picomole mg1 FW) followed by phosphatidylethanolamines
(PEs, ~270480 picomole mg1 FW) and phosphatidylglycerols
(PGs, ~4074 picomole mg1 FW) with the predominant species
in all these lipid classes being the 36 carbon and 34 carbon acyl
chains (e.g. PC 36:n and PC 34:n) that would be expected as
combinations of the most highly abundant C18 and C16 fatty
acyl chains as identied by GC-MS analysis (Tables S3, S8, S9).
Targeted lipid analysis was also able to quantify lower
abundance lipids such as LPCs and to allow the detection of
signicant differences in these classes of lipids and lipid species.
Proportional contributions of individual lipid species were
determined within a class as mol% and are presented in Fig. 3.
Analysis of phosphatidylcholines (PCs) in both genotypes
showed between 30 to nearly 50% of the 29 quantied PC
species were signicantly altered following exposure to salt.
In Sahara roots, nine PCs showed signicant mol% changes
with 6 decreased (PC 34 : 1, 34 : 2, 35 : 4, 36 : 1, 36 : 3, 36 : 4)
and three increased (PC 34 : 3, 36 : 5, 36 : 6) after salt. Clipper
roots showed signicant changes in 14 PCs (decreased: PC 33 : 1,
33 : 2, 33 : 3, 33 : 4, 35 : 4, 36 : 1, 36 : 2, 36:4; increased: PC 34 : 1,
34 : 2, 34 : 3, 36 : 0, 36 : 6, 38 : 2).

Functional Plant Biology

213

Sahara roots showed similar numbers of changes in

phosphatidylethanolamines (PEs) to Clipper roots with nine
PEs in Sahara showing signicant changes after salt (PE 32 : 1,
32 : 2, 34 : 1, 34 : 2, 35 : 4, 36 : 4 increased; PE 34 : 3, 36 : 6, 38 : 5
decreased) compared with seven in Clipper (PE 34 : 2, 36 : 6
increased; PE 32 : 1, 32 : 2, 33 : 3, 35 : 4, 36 : 4 decreased).
With the exception of the phosphatidylglycerol PG (34 : 3), all
the measured PGs were either unaltered or decreased following
salinity stress with the similar trend being observed in Clipper
roots.
In the low abundance lysophosphotidylcholine (LPC) class,
Clipper roots exposed to 100 mM NaCl salt for 5 weeks showed
a signicant decrease in LPC (15 : 0) when compared with the
Clipper control roots whereas in Sahara, the same salt exposure
resulted in a decrease in LPC (18 : 2) compared with the relevant
control. In both genotypes, a signicant increase in the second
most abundant LPC (LPC 18 : 3) was observed after salinity
stress. In the phosphatidylinositol (PI) lipid class, PIs containing
a total of 34 carbons, likely to contain combination of the
abundant C16 and C18 fatty acyl chains as determined by GCMS, showed the same signicant changes in both Clipper and
Sahara roots with PI (34 : 2) signicantly reduced and PI (34 : 3)
signicantly increased after salt. In the phosphatidylserine
(PS) lipid class, only Clipper showed signicant differences
following salt exposure with PS (34 : 2) showing a signicant
increase and PS (40 : 8) showing a signicant decrease to mol%
levels roughly that observed in the Sahara roots under both
treatment conditions.
Discussion
Overall, a few changes were observed in the total fatty acid
prole of roots of two barley genotypes with contrasting
responses to salinity, cv. Clipper and LR Sahara, and
signicant differences were also observed in the lipid proles
and specic phospholipid species detectable in the roots of
two barley genotypes, with cv. Clipper showing a greater
number of signicant differences after exposure to 100 mM
NaCl for 5 weeks than LR Sahara.
Previous work by Widodo et al. (2009) in studies of
responses of primary metabolites to salt stress in cv. Clipper
and LR Sahara, found that under the same experimental
conditions as described in our work (hydroponic growth for a
total of 6 weeks with exposure to 100 mM NaCl for 5 of
those weeks), cv. Clipper was more affected by salinity than
LR Sahara and although roots showed reduced amplitudes of
metabolite changes compared with leaves, signicant changes
in metabolite levels were observed in roots as a result of this
salt treatment. The more salt-sensitive cv. Clipper roots showed
numerous, high magnitude changes with most organic acids
dramatically decreased, as well as decreases in sugars, with the
exceptions being sorbitol and trehalose. Many amino acid
levels were also dramatically changed. This was in contrast to
LR Sahara roots where almost no changes in these metabolites
were detected after either 3 or 5 weeks of exposure to 100 mM
NaCl. Most notably, Clipper roots showed increases in proline,
sorbitol and the polyamine, putrescine whereas the less-salt
affected LR Sahara roots did not (Widodo et al. 2009).
A recent review by Mansour (2013), that stated proline,

214

Functional Plant Biology

sorbitol and polyamines are some of the compounds that

accumulate in salt tolerant plants and that are reported to
protect the plasma membrane under conditions of salinity
stress and may indicate that there is not always a positive
correlation between presence of the compounds and salinity
tolerance, a point also argued in an earlier review by
Greenway and Munns (1980).
Shelden et al. (2013) found that at the early barley seedling
stage (72 h after germination), LR Sahara showed a signicant
inhibition of root elongation whereas Widodo et al. (2009)
found that when grown under hydroponic conditions in the
longer term (100 mM NaCl exposure for 5 weeks) Sahara was
able to resume root growth despite showing earlier effects
under salinity stress whereas Clipper continued to reduce its
growth. In combination, these observations suggest that despite
early reductions in root growth in the presence of salt, Sahara
appears to have the capacity to recover and adapt under longterm salt stress imposed on more mature plants when grown
hydroponically, a potential tolerance mechanism which may
not have been apparent under shorter term growth conditions.
Phospholipid fatty acids can affect membrane uidity,
permeability and enzyme activity (Cooke and Burden 1990;
Spychalla and Desborough 1990). As part of the current work,
analysis of the fatty acid (FA) composition of the root lipid
extracts however, showed few signicant differences between
some of the FA levels present in cv. Clipper and LR Sahara
with and without salt-stress. Although some trends were
observed, no denitive differences were observed in changes
to the acyl chain lengths or in degrees of saturation or
unsaturation in response to salt. The only consistent difference
between the two genotypes after salt exposure was a decrease in
C16:1. This suggests that despite observations of changes in
other acyl-chain-containing metabolites such as more complex
lipid classes, the overall FA composition of roots is maintained
following salt exposure and that observations in changes in
specic phospholipids species may indicate re-location of the
associated fatty acid/acyl chains from elsewhere in the cell; for
example, the glycerolipids.
Surjus and Durand (1996) who used thin-layer
chromatography (TLC) in combination with GC to study the
phospholipid, total fatty acid and sterol proles of whole roots,
microsomal membranes and plasma membranes of soybean
seedlings grown hydroponically with a moderate concentration
of 25 mM NaCl, found that although no changes were detected
in whole soybean root tissues after salt treatment, increases in
fatty acid saturation could be observed in microsomal fractions
and to a lesser extent in the plasma membrane. This may suggest
that subcellular fractionation of barley root membranes in the
future may show changes in fatty acids not currently observed
in total root lipid extracts.
To compare overall root lipid proles from cv. Clipper and
LR Sahara with and without salinity stress, total root lipid

S. H. A. Natera et al.

extracts were compared by the technique of untargeted lipid

proling that utilises HPLC-ESI-QTOF-MS to separate and
analyse a wide range of lipid classes. This offers an approach
to compare lipid proles between samples when no or limited
prior knowledge is available about the lipid content or lipid
species of the sample in question. It can provide an overview,
inform and provide direction towards potential lipid classes to
be targeted for subsequent quantitative analysis.
Using this approach, root lipid proles from cv. Clipper and
LR Sahara showed a large proportion of changes in potential
lipids common to both genotypes as well as uniquely altered
in each genotype following salinity stress indicating possible
similarities in responses as well as genotype-specic changes
across a range of lipids such as phospholipids, glycerolipids
(neutral lipids such as DAGs and TAGs) and cardiolipins.
When compared with their respective controls, cv. Clipper
roots showed a larger number of signicant differences in their
lipid prole than LR Sahara roots after salt. As the phospholipids
represented the largest groups of potentially altered lipids,
many of which play roles in membrane lipid bilayer structures,
these were targeted for further quantitative analysis using
targeted lipid analysis. However, observations at the lipid
prole level indicate other lipid classes including glycerolipids
(such as diacylglycerols and triacylglycerols) and cardiolipins
may be differentially altered in barley roots following salinity
stress and represent additional lipid targets for further
investigation in the future.
Targeted lipidomics analysis using HPLC-ESI-QQQ-MS,
despite focusing on a relatively limited subset of lipids, has
the advantage of providing lipid class assignments based on
characteristic mass spectral fragmentation along with quantitative
data for comparison between samples (Blanksby and Mitchell
2010). As such, it provides a more selective, robust and
sensitive approach than untargeted analysis and provides
quantitative data that allows for a more detailed discussion
of the possible role of any measured lipid changes in the
biology of the system being studied.
The largest proportion of phospholipids measured in roots
using this approach were PCs which is similar to previous
observations in A. thaliana roots (Devaiah et al. 2006) with
PCs ranging from totals of 32 carbons up to 40 carbons with
the primary species containing totals of 34 and 36 carbons.
Sahara roots showed fewer changes in PCs after 5 weeks of
salt and those that did change showed a trend of increased
acyl chain unsaturation of the most abundant PCs, those that
contained 34 and 36 carbons in their acyl chains. In Clipper, a
larger proportion of PCs (48%) were altered following salt and
included decreases in PCs potentially containing odd-numbered
carbon chains which, to our knowledge, have not been measured
or reported in roots before. PCs are usually the most abundant
phospholipid classes in both plants and animals, as they are
the key lipid class in membrane bilayers, particularly in the

Fig. 3. Targeted (QQQ) lipid analysis showing mol% of individual phospholipids measured in barley root lipid extracts from the barley genotypes Clipper
and Sahara after 5 weeks of salinity stress (100 mM NaCl) compared with their respective controls as measured by HPLC-ESI-QQQ-MS/MS MRM (multiple
reaction monitoring scans). Abbreviations: Clip C, Clipper control; Clip S, Clipper salt-treated; Sah C, Sahara control; Sah S, Sahara salt-treated. LPC,
lysophosphatidylcholine; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol;
PS, phosphatidylserine. Values are means of six replicate samples for each treatment group (n = 6)  s.e. Lipid species are annotated as follows: lipid class
designation (total number of carbon atoms in the acyl chains: total number of double bonds in the fatty acid chains), e.g. PC (32 : 0).

Lipidomics of barley roots in response to salt

LPC (15:0)

3
2

LPC (18:1)

LPC (18:2)

10.0

7.5

5.0

2.5

LPC (18:3)

40

LPC (24:4)

20
10
0

LPC(17:0)

PE (32:1)

PE (32:2)

PE (33:1)
0.125

2.0

1.5

0.2

1.0

0.1

0.050

PE (33:3)

PE (34:2)

30

PE (34:3)

1.0

0.4
0.2
0

PE (35:3)

20

10

PE (35:4)

5
0

PE (36:3)

0.20

2.0

0.15

1.5

0.10

PE (36:4)

30

0.05

0.5

PE (38:5)
0.3

0.6

20

PE (38:4)
0.8

PE (36:5)

12

1.0

0.03

10

PG (32:0)

PG (34:1)

1.5

0.2

1.0

0.1

0.5

0.6

40
0.4

Sa
h

h
Sa

h
Sa

lip

Fig. 3. (Continued).

0
S

0
S

0.5

Sa
h

Sa

lip
C

C
lip

lip

lip

S
Sa
h
C
Sa
h
S

0
C

1.0

10

1.5

0.2

C
lip

20

20

Sa

30

h
Sa

PG (36:5)
2.0

PG (36:4)

lip

40

PG (34:3)
60

C
lip

PG (34:2)

C
lip

Sa

0.2

C
lip

0.4

15

0.09
0.06

20

10

0.5

0.025
0

PE (34:1)
3

0.6

0.075

0.5

PE (33:2)

Mol %

0.100

0.3

50

30

0.4

10

40

PE (36:6)

215

20

LPC (16:0)

Functional Plant Biology

Functional Plant Biology

S. H. A. Natera et al.

PC (32:1)

PC (32:2)

0.08

0.20

0.06

0.15

0.04

0.10

0.02

0.05

PC (32:3)

PC (33:1)
1.5

0.10
0.08

PC (33:3)

0.05

0.04

0.5

PC (34:0)

0.6

1.0

0.03

0.05

0.5

15

10

0.3

PC (34:4)

20
15

0.10

0.06

PC (34:3)

PC (35:1)

PC (35:2)

0.03

0.3

0.02

0.2

0.01

0.1

PC (35:3)
0.20

0.2

0.15

10

0.10
0.1

5
0

PC (35:4)

PC (36:0)

0.10

0.20

PC (36:5)

15

15

10

10

PC (38:2)

1.5

0.3
0.2

0.5
0.1

PC (38:5)

PC (39:2)

0.15

0.06

0.2

0.10

0.04

0.1

0.05

0.02

PC (41:6)
0.020
0.015

0.005

Fig. 3. (Continued).

Sa

h
Sa

C
lip

C
lip

h
Sa

h
Sa

lip

0
C
lip

lip
C
C
lip
S
Sa
h
C
Sa
h
S

Sa
h

h
Sa

0.010

C
lip

PC (38:3)
0.4

1.0

PC (38:4)

PC (36:6)
9

0.3

0.50
2

20

0.25

25

0.05

PC (36:4)

PC (36:3)
4

0.10
0.2

20

0.75

0.15

0.4

0.05

25

PC (36:2)

0.25

0.6

PC (36:1)

0.15

0.05

C
lip

Mol %

PC (34:2)

1.5

PC (34:1)

0.15
0.09

1.0

PC (33:4)

0.9

PC (33:2)

0.12

lip
C
C
lip
S
Sa
h
C
Sa
h
S

216

Lipidomics of barley roots in response to salt

plasma membrane. Alterations in PCs may have implications in

the structure and function of the plasma membrane and other
organelle membranes in the cell either at the cause or effect
level. As the most abundant phospholipid, changes in PCs
could potentially lead to changes in membrane uidity and
permeability as well as having effects on integral proteins
embedded in the membrane (Upchurch 2008). Increasing
levels of unsaturation in PC acyl chains may potentially be
linked to increased membrane uidity (van Meer et al. 2008).
PEs are the second most abundant phospholipids in plants
and animals and are also key components of membrane
bilayers. Despite showing similar numbers of changes in both
genotypes, more PEs appeared to increase after salinity stress
in Sahara roots compared with Clipper roots, in which more of
the changes were decreases.
PGs are the major phospholipid in thylakoid membranes
of higher plants and can be used as a precursor of cardiolipins
located on the inner mitochondrial membrane that are required
for proper functioning of the oxidative phosphorylation
enzymes (Ren et al. 2014). The low levels of PGs measured in
this study are consistent with the non-photosynthetic nature
of root tissue however, signicant changes were observed
in the PGs detectable in barley roots. The overall observation
of unchanged or signicantly decreased PGs following salt
exposure may indicate reduced levels of plastid biogenesis
under these conditions that also may be indicative of stress
responses (Narasimhan et al. 2013).
The relationship between the phospholipid species present in
a membrane and the levels of saturation or unsaturation of
their acyl chains, along with other lipids such as sphingolipids
and sterols not targeted in this work, can combine to give
complex lipid phase behaviours that can affect membrane
uidity and potential permeability (van Meer et al. 2008).
Membrane permeability, in particular of the plasma membrane,
may also act as an indicator of salt tolerance in plants (Mansour
2013).
Along with the reduced growth and affected polar metabolite
proles previously demonstrated by Widodo et al. (2009), cv.
Clipper roots showed more changes in phospholipids and
overall lipid proles than their LR Sahara counterparts in
response to salinity stress. In combination, this seems to be
consistent with previous ndings that in salt-tolerant plants,
membrane integrity or at least plasma membrane integrity is
more highly maintained (DuPont et al. 1988; Brown and
DuPont 1989; Wu et al. 2005; Chalbi et al. 2015). Salinity
tolerance may be conferred by several different mechanisms
including changes in membrane permeability not only directly
by changes in membrane lipid composition but by changes in
presence and activity of potential ion transporters, where
membrane composition may also play a potential role in the
regulation of such ion transporters (Carruthers and Melchior
1986; Lee 2004). Further investigation via closer analysis
of the lipidome of root subcellular membrane fractions, in
particular, the plasma membrane and membrane modelling
(Sommer 2013) to examine the possible effects on membrane
structure and uidity based on these observed changes and
differences would be of interest as would investigation of
possible ow-on effects there might be on membraneassociated protein and enzyme function (Flowers et al. 2015).

Functional Plant Biology

217

In summary, the untargeted and targeted mass-spectrometrybased lipidomics technologies described in the current work
were applied to the analysis of the lipidome of barley roots
from genotypes with differing responses to salinity and a
range of alterations could be seen in the lipids, particularly
phospholipids, following 5 weeks of exposure to 100 mM NaCl.
Our current knowledge on the role of individual lipid
species in planta under environmental stress is lacking, both in
roots and shoots; however, the present work has demonstrated
the importance of lipids in stress adaptation and tolerance. The
exploration of genetic diversity in lipid proles in a range of
genotypes with contrasting salinity adaptation and tolerance
levels will shed more light into potential functions of
individual lipid species or lipid classes in salinity tolerance. In
addition, combining the described lipidomics approaches with
available genetic resources of mapping populations (Karakousis
et al. 2003) will provide pathways for the identication of
underlying genetic and molecular mechanisms potentially
involved in salinity adaptation and tolerance.
Acknowledgements
The authors thank Ms Nirupama Jayasinghe (Metabolomics Australia,
School of BioSciences, The University of Melbourne) for providing
excellent technical assistance with the GC-MS and LC-MS analysis,
Dr Stuart Roy (Australian Centre for Plant Functional Genomics, The
University of Adelaide) for providing the barley seeds and Lisa
Pasipanodya for assistance with plant growth. The authors are grateful to
the Victorian Node of Metabolomics Australia, which is funded through
Bioplatforms Australia Pty Ltd, a National Collaborative Research
Infrastructure Strategy (NCRIS), 5.1 Biomolecular platforms and
informatics investment and co-investment from the Victorian State
Government and The University of Melbourne.

References
Adem GD, Roy SJ, Zhou M, Bowman JP, Shabala S (2014) Evaluating
contribution of ionic, osmotic and oxidative stress components towards
salinity tolerance in barley. BMC Plant Biology 14(1), 113. doi:10.1186/
1471-2229-14-113
Blanksby SJ, Mitchell TW (2010) Advances in mass spectrometry for
lipidomics. Annual Review of Analytical Chemistry (Palo Alto, Calif.)
3, 433465. doi:10.1146/annurev.anchem.111808.073705
Brown DJ, DuPont FM (1989) Lipid composition of plasma membranes
and endomembranes prepared from roots of barley (Hordeum vulgare
L.): effects of salt. Plant Physiology 90, 955961. doi:10.1104/pp.
90.3.955
Carruthers A, Melchior DJ (1986) How bilayer lipids affect membrane
protein activity. Trends in Biochemical Sciences 11(8), 331335.
doi:10.1016/0968-0004(86)90292-6
Chalbi N, Martnez-Ballesta MC, Youssef NB, Carvajal M (2015)
Intrinsic stability of Brassicaceae plasma membrane in relation to
changes in proteins and lipids as a response to salinity. Journal of
Plant Physiology 175, 148156. doi:10.1016/j.jplph.2014.12.003
Cooke DT, Burden RS (1990) Lipid modulation of plasma membranebound ATPases. Physiologia Plantarum 78(1), 153159. doi:10.1111/
j.1399-3054.1990.tb08730.x
Devaiah SP, Roth MR, Baughman E, Li M, Tamura P, Jeannotte R, Welti R,
Wang X (2006) Quantitative proling of polar glycerolipid species
from organs of wild-type Arabidopsis and a PHOSPHOLIPASE Da1
knockout mutant. Phytochemistry 67(17), 19071924. doi:10.1016/
j.phytochem.2006.06.005

218

Functional Plant Biology

DuPont FM, Tanaka CK, Hurkman WJ (1988) Separation and

immunological characterization of membrane fractions from barley
roots. Plant Physiology 86, 717724. doi:10.1104/pp.86.3.717
Fahy E, Cotter D, Byrnes R, Sud M, Maer A, Li J, Nadeau D, Zhau Y,
Subramaniam S (2007a). Bioinformatics for lipidomics. In Methods
in enzymology. (Ed. HA Brown) pp. 247273. (Academic Press: New
York)
Fahy E, Sud M, Cotter D, Subramaniam S (2007b) LIPID MAPS online
tools for lipid research. Nucleic Acids Research 35, W606W612.
doi:10.1093/nar/gkm324
Fahy E, Subramaniam S, Murphy R, Nishijima M, Raetz C, Shimizu T,
Spener F, van Meer G, Wakelam M, Dennis EA (2009) Update of
the LIPID MAPS comprehensive classication system for lipids.
Journal of Lipid Research 50, S9S14. doi:10.1194/jlr.R800095JLR200
FAO (2012) Food and Agriculture Organization of the United Nations
(FAO) statistical databases. Available at http://faostat.fao.org [Veried
20 August 2015]
Flowers TJ, Munns R, Colmer TD (2015) Sodium chloride toxicity and the
cellular basis of salt tolerance in halophytes. Annals of Botany 115(3),
419431. doi:10.1093/aob/mcu217
Genc Y, McDonald GK, Tester M (2007) Reassessment of tissue Na+
concentration as a criterion for salinity tolerance in bread wheat. Plant,
Cell & Environment 30, 14861498. doi:10.1111/j.1365-3040.2007.
01726.x
Greenway H, Munns R (1980) Mechanisms of salt tolerance in Nonhalophytes.
Annual Review of Plant Physiology 31(1), 149190. doi:10.1146/annurev.
pp.31.060180.001053
Hill CB, Bacic A, Roessner U (2014) LC-MS proling to link metabolic
and phenotypic diversity in plant mapping populations. In Mass
spectrometry in metabolomics. pp. 2941. (Springer: New York)
Horn PJ, Chapman KD (2011) Organellar lipidomics. Plant Signaling &
Behavior 6(10), 15941596. doi:10.4161/psb.6.10.17133
Horn PJ, Chapman KD (2012) High-resolution measurements in
plant biology: lipidomics in tissues, cells and subcellular
compartments. The Plant Journal 70, 6980. doi:10.1111/j.1365313X.2011.04868.x
Hu CH, van Dommelen J, van der Heijden R, Spijksma G, Reijmers TH,
Wang M, Slee E, Lu X, Xu G, van der Greef J, Hankemeier T (2008)
RPLC-Ion-Trap-FTMS method for lipid proling of plasma: method
validation and application to p53 mutant mouse model. Journal of
Proteome Research 7, 49824991. doi:10.1021/pr800373m
Karakousis A, Barr AR, Kretschmer JM, Manning S, Jefferies SP,
Chalmers KJ, Islam AKM, Langridge P (2003) Mapping and QTL
analysis of the barley population Clipper Sahara. Crop and Pasture
Science 54(12), 11371140. doi:10.1071/AR02180
Lee AG (2004) How lipids affect the activities of integral membrane
proteins. Biochimica et Biophysica Acta (BBA) Biomembranes
1666(12), 6287. doi:10.1016/j.bbamem.2004.05.012
Lee YJ, Perdian DC, Song Z, Yeung ES, Nikolau BJ (2012) Use of mass
spectrometry for imaging metabolites in plants. The Plant Journal 70(1),
8195. doi:10.1111/j.1365-313X.2012.04899.x
Mansour MMF (2013) Plasma membrane permeability as an indicator of
salt tolerance in plants. Biologia Plantarum 57(1), 110. doi:10.1007/
s10535-012-0144-9
Meijer HJ, Munnik T (2003) Phospholipid-based signaling in plants.
Annual Review of Plant Biology 54, 265306. doi:10.1146/annurev.
arplant.54.031902.134748
Mittler R (2006) Abiotic stress, the eld environment and stress
combination. Trends in Plant Science 11(1), 1519. doi:10.1016/
j.tplants.2005.11.002
Munns R, Tester M (2008) Mechanisms of salinity tolerance. Annual
Review of Plant Biology 59, 651681. doi:10.1146/annurev.arplant.59.
032607.092911

S. H. A. Natera et al.

Narasimhan R, Wang G, Li M, Roth M, Welti R, Wang X (2013)

Differential changes in galactolipid and phospholipid species in
soybean leaves and roots under nitrogen deciency and after
nodulation. Phytochemistry 96, 8191. doi:10.1016/j.phytochem.2013.
09.026
Okazaki Y, Saito K (2014) Roles of lipids as signaling molecules and
mitigators during stress response in plants. The Plant Journal 79(4),
584596. doi:10.1111/tpj.12556
Okazaki Y, Kamide Y, Hirai MY, Saito K (2013) Plant lipidomics based
on hydrophilic interaction chromatography coupled to ion trap time-ofight mass spectrometry. Metabolomics 9(1), 121131. doi:10.1007/
s11306-011-0318-z
Olmstead ILD, Hill DRA, Dias DA, Jayasinghe NS, Callahan DL, Kentish SE,
Scales PJ, Martin GJO (2013) A quantitative analysis of microalgal lipids
for optimization of biodiesel and omega-3 production. Biotechnology
and Bioengineering 110(8), 20962104. doi:10.1002/bit.24844
Pluskal T, Castillo S, Villar-Briones A, Oreic M (2010) MZmine 2:
Modular framework for processing, visualizing, and analyzing mass
spectrometry-based molecular prole data. BMC Bioinformatics 11,
395. doi:10.1186/1471-2105-11-395
Ren M, Phoon CKL, Schlame M (2014) Metabolism and function of
mitochondrial cardiolipin. Progress in Lipid Research 55, 116.
doi:10.1016/j.plipres.2014.04.001
Samarakoon T, Shiva S, Lowe K, Tamura P, Roth MR, Welti R (2012)
Arabidopsis thaliana membrane lipid molecular species and their mass
spectral analysis. High-Throughput Phenotyping in Plants: Methods
and Protocols 918, 179268.
Shelden MC, Roessner U, Sharp RE, Tester M, Bacic A (2013) Genetic
variation in the root growth response of barley genotypes to salinity
stress. Functional Plant Biology 40(5), 516530. doi:10.1071/FP12290
Shi BJ, Sutton T, Collins NC, Pallotta M, Langridge P (2010) Construction
of a barley bacterial articial chromosome library suitable for cloning
genes for boron tolerance, sodium exclusion and high grain zinc
content. Plant Breeding 129(3), 291296. doi:10.1111/j.1439-0523.
2009.01762.x
Shiva S, Vu HS, Roth MR, Zhou Z, Marepally SR, Nune DS, Lushington
GH, Visvanathan M, Welti R (2013) Lipidomic analysis of plant
membrane lipids by direct infusion tandem mass spectrometry. Plant
Lipid Signaling Protocols 1009, 7991.
Sommer B (2013) Membrane packing problems: a short review on
computational membrane modeling methods and tools. Computational
and Structural Biotechnology Journal 5(6), 113. doi:10.5936/csbj.20
1302014
Spychalla JP, Desborough SL (1990) Fatty acids, membrane permeability,
and sugars of stored potato tubers. Plant Physiology 94(3), 12071213.
doi:10.1104/pp.94.3.1207
Surjus A, Durand M (1996) Lipid changes in soybean root membranes in
response to salt treatment. Journal of Experimental Botany 47(1),
1723. doi:10.1093/jxb/47.1.17
Tavakkoli E, Rengasamy P, McDonald GK (2010) High concentrations of
Na+ and Cl ions in soil solution have simultaneous detrimental effects
on growth of faba bean under salinity stress. Journal of Experimental
Botany 61(15), 44494459. doi:10.1093/jxb/erq251
Upchurch RG (2008) Fatty acid unsaturation, mobilization, and regulation
in the response of plants to stress. Biotechnology Letters 30, 967977.
doi:10.1007/s10529-008-9639-z
van Meer G (2005) Cellular lipidomics. EMBO Journal 24, 31593165.
doi:10.1038/sj.emboj.7600798
van Meer G, Voelker DR, Feigenson GW (2008) Membrane lipids: where
they are and how they behave. Nature Reviews. Molecular Cell Biology
9(2), 112124. doi:10.1038/nrm2330
Walia H, Wilson C, Condamine P, Liu X, Ismail AM, Close TJ (2007)
Large-scale expression proling and physiological characterization of
jasmonic acid-mediated adaptation of barley to salinity stress. Plant,

Lipidomics of barley roots in response to salt

Functional Plant Biology

Cell & Environment 30(4), 410421. doi:10.1111/j.1365-3040.2006.

01628.x
Wang X (2002) Phospholipase D in hormonal and stress signalling.
Current Opinion in Plant Biology 5, 408414. doi:10.1016/S13695266(02)00283-2
Weber H (2002) Fatty acid-derived signals in plants. Trends in Plant Science
7(5), 217224. doi:10.1016/S1360-1385(02)02250-1
Welti R, Wang X (2004) Lipid species proling: a high-throughput
approach to identify lipid compositional changes and determine the
function of genes involved in lipid metabolism and signaling. Current
Opinion in Plant Biology 7(3), 337344. doi:10.1016/j.pbi.2004.03.011
Wenk MR (2005) The emerging eld of lipidomics. Nature Reviews. Drug
Discovery 4(7), 594610. doi:10.1038/nrd1776
Widodo , Patterson JH, Newbigin E, Tester M, Bacic A, Roessner U (2009)
Metabolic responses to salt stress of barley (Hordeum vulgare L.)
cultivars, Sahara and Clipper, which differ in salinity tolerance.

219

Journal of Experimental Botany 60(14), 40894103. doi:10.1093/jxb/

erp243
Wu J, Seliskar DM, Gallagher JL (2005) The response of plasma membrane
lipid composition in callus of the halophyte Spartina patens (Poaceae)
to salinity stress. American Journal of Botany 92(5), 852858.
doi:10.3732/ajb.92.5.852
Xia J, Sinelnikov IV, Han B, Wishart DS (2015) MetaboAnalyst 3.0 making
metabolomics more meaningful. Nucleic Acids Research 43(W1),
W251W257. doi:10.1093/nar/gkv380
Yoon JY, Hamayun M, Lee SK, Lee IJ (2009) Methyl jasmonate
alleviated salinity stress in soybean. Journal of Crop Science and
Biotechnology 12(2), 6368. doi:10.1007/s12892-009-0060-5
Zhou X-R, Callahan DL, Shrestha P, Liu Q, Petrie JR, Singh SP (2014)
Lipidomic analysis of Arabidopsis seed genetically engineered to
contain DHA. Frontiers in Plant Science 5, 419. doi:10.3389/fpls.
2014.00419

www.publish.csiro.au/journals/fpb