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Feature Review

Plant single-cell and single-cell-type


metabolomics
Biswapriya B. Misra1, Sarah M. Assmann2, and Sixue Chen1,3
1

Department of Biology, Genetics Institute, Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL
32610, USA
2
Department of Biology, Penn State University, 208 Mueller Laboratory, University Park, PA 16802, USA
3
Interdisciplinary Center for Biotechnology Research, University of Florida, Gainesville, FL 32610, USA

In conjunction with genomics, transcriptomics, and proteomics, plant metabolomics is providing large data sets
that are paving the way towards a comprehensive and
holistic understanding of plant growth, development,
defense, and productivity. However, dilution effects
from organ- and tissue-based sampling of metabolomes
have limited our understanding of the intricate regulation of metabolic pathways and networks at the cellular
level. Recent advances in metabolomics methodologies,
along with the post-genomic expansion of bioinformatics knowledge and functional genomics tools, have
allowed the gathering of enriched information on individual cells and single cell types. Here we review progress, current status, opportunities, and challenges
presented by single cell-based metabolomics research
in plants.
Metabolomics at cellular resolution
Metabolites add an important extra dimension to the
information flow from DNA to RNA to protein. With the
advent of high-end instrumentation and an accelerated
understanding of the roles of metabolites in organismal
functions [1], our appreciation of the importance of metabolites is increasing. By definition, the metabolome is the
entire set of small molecules (molecular mass less than
2,000 Da) or metabolites in a given biological sample.
Metabolomics is the unbiased analytical assessment of
the metabolome, whereas metabolite/metabolic profiling
deals with the detection and quantification of a group of
metabolites using a particular analytical approach [2]. The
total number of metabolites found in plants is currently
estimated at 200,000, with around 7,00015,000 being
found in any individual species [3,4], 3,0005,000 of which
reside in leaves [5,6]. These large groups of structurally
diverse, temporally and spatially dynamic compounds pose
great challenges for modern analytical technologies [7]
compared with the comparatively straightforward polymeric assemblies of four nucleotides as DNA molecules.
In addition, species specificity in the distribution of specialized metabolites, rapid endogenous turnover rates, an
Corresponding author: Chen, S. (schen@ufl.edu).
Keywords: metabolomics; mass spectrometry; single cell; single cell type; systems
biology.
1360-1385/
2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tplants.2014.05.005

inability to amplify metabolites, the limited ability to


capture more than a snapshot of the metabolites in an
organism at any one time, the suboptimal quality of extraction methods, and the dynamic ranges of abundance,
detection capabilities, ionization and polarizability render
metabolomics far more difficult than other omics. It is no
doubt that metabolomics studies are of essential importance for a comprehensive understanding of cellular functions [8,9], and metabolomics has become important in
studying the genetic regulation of metabolite synthesis
and function [10]. Large-scale discovery and quantitation
of metabolites is imperative for comprehensive understanding of plant signaling and biochemical processes.
In addition, many phytochemicals have immense significance in food, medicine, and agriculture.
Alongside tissue-, organ-, and organism-level metabolome studies, tools and techniques have been developed to
overcome the averaging effect that is inevitable in studies
on populations of mixed cell types. Indeed, advances in the
isolation of both single cells and populations of an individual cell type have made metabolomics at the cellular level a
reality, thus enhancing unbiased detection and quantification of metabolites at this basic level of biological organization [11]. Cell types have been defined on the basis of
their appearance, location, expression of specific marker
genes, or performance of specific functions [12]. To date, the
choice of cell systems to study has been dictated largely by
the ease of isolation, handling, purity and homogeneity of
preparations, state of differentiation, and importance to
the plants physiological status. Here, we define single-cell
metabolomes as those derived from one individual cell
(e.g., a single epidermal cell), whereas single-cell-type
metabolomes are defined as those derived from a population of cells representing a common origin and physiological role (e.g., trichomes or guard cells).
Higher plants are multicellular organisms: for example,
in rice 40 different cell types are documented [13]. To date,
enriched or pure preparations of many cell types from
different plant species have been used for single-cell or
single-cell-type metabolomics studies (Table 1, Figure 1).
Excellent topical reviews have appeared that summarize
substantial progress in the areas of single-cell genomics
[14], transcriptomics [15], and proteomics [16] of plants. In
this review, we present recent advances in plant single-cell
and single-cell-type metabolomics. We highlight the sampling tools, techniques, and various analytical platforms,
Trends in Plant Science, October 2014, Vol. 19, No. 10

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Feature Review

Trends in Plant Science October 2014, Vol. 19, No. 10

Table 1. Recent studies highlighting the analytical tools used and results obtained in single cell and single cell-type metabolomics
Species

Material

Subcellular metabolomics
Vacuoles from mesophyll
Hordeum vulgare
cell protoplasts
Single-cell metabolomics
Petal cell
Torenia hybrida
Single cell
Closterium acerosum
Single leaf, stem, petal cell
Pelargonium zonale
Epidermal single cell of bulbs
Allium cepa, Narcissus
pseudonarcissus
Individual dark glands from
Arabidopsis thaliana,
Hypericum perforatum, petals, leaves; glandular
trichomes
Hypericum reflexum
Single-cell-type metabolomics
Leaf and flower secretory
Dilatris pillansii
cavities
Leaf subdermal secretory
Eucalyptus spp.
cavities
Leaf epidermome
Catharanthus roseus
Phloem parenchyma cells
Picea abies
Stone cells/sclereids
P. abies
Phloem latex (sieve tubes)
Cucurbita maxima

Analytical
approach a

Metabolites
identified

Class of metabolites

Refs

GC-MS,
UPLC-FT-MS

259

Amino acids, organic acids,


sugars, specialized metabolites

[45]

Nano-HPLC-MS
MALDI-MS
Nano-ESI-MS
AP-ESI-MS

5
4
22
32, 22

[33]
[79]
[36]
[34]

LDI-ToF-MS

15

Anthocyanins
Central metabolites
Monoterpenoids
Specialized metabolites,
oligosaccharides
Naphthodianthrones, flavonoids

Cryogenic
NMR, HPLC
GC, GC-MS

Methoxyphenylphenalenones

[50]

24

Mono- and sesqui-terpenoids

[51]

LC-ESI-MS
Cryogenic NMR
Cryogenic NMR, MS
GC-ToF-MS,
HPLC, FIE-MS
GC-MS,
UPLC-QToF-MS

2
2
2
80

Oleanolic and ursolic acid


Stilbene glucosides, flavonoids
Phenolic glycosides
Amino acids, sugars

[52]
[43]
[54]
[55]

634

Amino acids, sugars, sugar alcohols,


fatty acids, flavonoids, organic acids,
nucleosides, phenolics, glucosinolates,
saponins, alkaloids
Sugars, organic acids,
amino acids
Non-polar (sterols, alkanes)
and polar (sugars, sugar
alcohols, amino acids)
Organic acids, amino acids,
sugars
Terpenoid, sterols, fatty acids,
carotenoids, oxygen heterocyclics
Phytohormones, signaling molecules,
phenolics, flavonoids, amino acids
Terpenoids
Terpenoid, flavonoids, fatty
acids, alkaloids, acyl sugars
Acylated molecules, flavonoids,
glycosides,
Mono- and sesqui-terpenoids
Cannabinoids
Amino acids, fatty acids and alcohols,
alkanes, lipids, N-compounds, organic
acids, polyhydroxy acids, polyols,
sugars, sugar conjugates,
phenylpropanoids
Glucosinolates
Sesquiterpenoids (colquhounoids)

[56]

[70]

[41]

Glycine max

Root hairs

Lilium longiflorum

Pollen grains

GC-ToF-MS

252

Gossypium hirsutum

Fiber cells

GC-MS

86

G. hirsutum

Fiber cells

GC-MS

27

Citrus paradisi

GC, GC-MS,
UPLC-QToF-MS
LC-(MRM)-MS/MS

28
85

Lycopersicon hirsutum
Solanum spp.

Epithelial and parenchyma


cells
Guard cell and mesophyll
cell protoplasts
Glandular trichomes
Trichomes

GC-MS
LC-MS

7
119

Solanum lycopersicum

Glandular trichomes

LC-ToF-MS

32

Artemisia annua
Cannabis sativa
A. thaliana

Glandular trichomes
Trichome types
Epidermal trichomes,
basal/pavement cells

GC-MS
LC-MS, NMR
GC-ToF-MS

12
9
117

A. thaliana
Colquhounia coccinea

Glandular trichomes
Peltate glandular trichomes

13
3

Nicotiana attenuate

Glandular trichomes

Ocimum basilicum

Peltate and capitate


glandular trichomes
Epidermal cell layer,
palisade mesophyll cells,
vascular bundle
Endodermis, epidermis,
columella, cortex, stele

UPLC-ESI-QToF-MS
UPLC-MS/MS,
X-ray diffraction
1
H-NMR,
LC-QToF-MS
GC-MS, HPLC

15

Nicotine, phaseoloidin,
acyl sugars, fatty acids
Terpenoids, phenylpropenes

MALDI-ToF-MS

18

Cell wall polysaccharides

[80]

GFP-FACS,
UPLC-QToF-MS

50

Glucosinolates, phenylpropanoids,
dipeptides

[81]

A. thaliana

A. thaliana

A. thaliana
a

>2

[57]
[59]

[60]
[62]
[23]
[66]
[65]
[67]
[68]
[69]
[63]

[64]
[42]

[71]

Abbreviations: AP, atmospheric pressure; ESI, electrospray ionization; FACS, fluorescence activated cell sorting; FIE, flow injection electrospray; FT, Fourier transform; GC,
gas chromatography; HPLC, high performance liquid chromatography; LC, liquid chromatography; LDI, matrix-free laser desorption/ionization; MALDI, matrix-assisted
laser desorption/ionization; MRM, multiple reaction monitoring; MS, mass spectrometry; MS/MS, tandem mass spectrometry; NMR, nuclear magnetic resonance; QToF,
quadrupole time of flight; ToF, time of flight; UPLC, ultra performance liquid chromatography.

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Feature Review

Trends in Plant Science October 2014, Vol. 19, No. 10

Epidermal
cells
(pavement,
basal)
Trichomes
(glandular/
nonglandular)

Secretory
cavies

Mesophyll
cells

Epidermal
parenchyma

Petal
cells

Pollen

Secretory
cavies

Guard cells

Flower and Fruit

Leaf

Phloem
parenchyma

MS

Spectra

Xylem
bers

Stele

Laser microdissecon
Micromanipulaon
Mechanical isolaon
Protoplasng
Cell sorng

Metabolite

Metabolomics

Pericycle
cells

Sclereids

Trichomes
(glandular,
nonglandular)

Sieve tubes
(Phloem
exudates)

Stem

Plant

Single cell types

Root
hairs

Cortex

Endodermis

Columella

Single cell

Root

Subcellular
TRENDS in Plant Science

Figure 1. Overview of current metabolomics studies of plant single cells and single cell types. Single cell types are obtained from plant parts such as leaves, fruits, flowers,
stems, and roots by means of laser microdissection, micromanipulation, mechanical isolation, protoplasting, and cell sorting. Subcellular organelles and single plant cells
are analyzed using microsampling, mass spectrometry imaging (MSI), nano-electrospray ionization (ESI) tip, and other means, including differential centrifugation.
Chromatography, mass spectrometry (MS), and nuclear magnetic resonance (NMR) tools have allowed the analysis of cellular metabolomes.

and summarize recent discoveries regarding metabolome


content and related cellular processes that have been made
using these approaches.
Technologies in sampling single cells and single cell
types
Major methods of sample preparation for plant metabolomic studies on single cells and single cell types are summarized in Box 1. It is rightly understood that single-cell or
cell-type-specific experiments performed on isolated cells
may not represent the actual metabolic status of a cell in the
plant [11,17], partly owing to fast metabolic turnover on a
time scale of seconds or less, and the disruption of proximity
to other cell types and intercellular communication [18,19].

Accordingly, recent efforts have also focused on methods


whereby single cells can be obtained or extracted in situ
[20]. These methods include micropipetting methods for
isolating the contents of specific cells (e.g., epidermal
pavement cell and sieve tube members) [12], laser microdissection (LMD), laser microdissection and pressure
catapulting (LMPC), and laser capture microdissection
(LCM) [21] (Box 1).
Isolated populations of single cell types are obtained
primarily by protoplasting and suspension cell culture [22].
Protoplasting has the potential to provide large quantities
of cells (e.g., mesophyll cells [2326]) but is challenging for
those cell types that are uncommon, (e.g., guard cells) or
surrounded by highly lignified cell walls (e.g., sclereids).

Box 1. Sampling methods for single cells and single cell types in plants
LMD
Laser microdissection (LMD) is a popular tool for direct time-resolved
selection, isolation, and sampling of cells from fixed or frozen tissues
[12]. A recent review of the LMD workflow, sample preparation,
microdissection, post-harvest treatment, and microdissection steps
discusses applications in metabolic profiling of a wide range of cells
and tissues, including stem, leaves, roots, flowers, and seeds [82].
The challenges associated with LMD include its labor intensiveness
and the limited quantities of proteins and metabolites available for
proteomic and metabolomic analysis [12,8386].
LMPC and LCM
Laser microdissection and pressure catapulting (LMPC) and laser
capture microdissection (LCM), both of which use a laser to excise
single cells or microareas from intact tissues, are becoming popular
for plant cell and tissue sampling [84]. For instance, a combination of
LMD-assisted isolation of 4,000 single cells followed by enzymatic
digestion of polymeric cell-wall materials from Arabidopsis enabled
identification of several sugars [80].
FACS
Fluorescence activated cell sorting (FACS) is used to obtain specific
cell types: for example, those identified from root developmental

zones [12,81] by transgene-labelled nuclei or by immunobead-based


collection and microfluidic sorting based methods that exploit
intrinsic cell properties [12,87]. Recently, FACS was used to dissect
green fluorescence protein (GFP)-marked core cell types from
Arabidopsis roots for metabolomics, leading to the identification of
about 50 metabolites, primarily glucosinolates, phenylpropanoids,
and dipeptides [81].
Protoplasting
Protoplasts, cell-wall-less single cell types, are obtained by enzymatic
removal of the cell wall from plant cells and tissues. Over the past 50
years, protoplasting has offered a wide range of applications,
including in single-cell omics studies [2326,4648].
Cell suspension culture
A cell suspension culture is a population of undifferentiated plant
cells growing in liquid medium containing nutrients and hormones,
particularly auxin and cytokinin. The cells are originally derived from
plant tissues that are dedifferentiated into callus and subsequently
become individual cells in the liquid medium. Cell suspension
cultures as model systems are useful for studying plant cell division,
the cytoskeleton, hormone signaling, membrane transport, metabolite production, and responses to environmental factors.
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Feature Review
Major criticisms associated with protoplasting include loss
of cell-wall-related information and perturbation of the
original cell state. Interestingly, recent findings suggest
that only a small subset of transcripts are upregulated or
activated during protoplasting, and the overall global gene
expression profile remains unaltered [11]. Please refer to
Box 1 and Single-cell-type metabolomics applications for
suspension cell culture.
Technologies for analyzing metabolomes of single cells
and single cell types
High-coverage metabolomics is challenging, and more so in
single-cell and single-cell-type studies owing to the large
dynamic range (from a few molecules to millions of molecules per cell) and lack of any method analogous to PCR
that could amplify metabolites. However, multidisciplinary efforts have immensely expanded the inventory of
detectable chemical entities. Following sampling and metabolite extraction, several methods are available for metabolite profiling, including nuclear magnetic resonance
(NMR) spectroscopy, mass spectrometry (MS) [27], and
specialized amperometry or fluorescence methods [28].
Although the physical foundation for NMR was laid as

Trends in Plant Science October 2014, Vol. 19, No. 10

early as the 1930s, NMR spectroscopy in metabolite profiling of plant cells was first reported in 1988 [29]. NMR
remains the gold standard for de novo structural elucidation but has low sensitivity and limited applications for
large-scale profiling. MS is usually the analytical tool of
choice [30]. To enhance the analytic capability, front-end
separation technologies such as gas chromatography (GC),
liquid chromatography (LC), ultra performance liquid
chromatography (UPLC), and capillary electrophoresis
(CE) are often used in conjunction with MS. Metabolite
profiling by GC-MS dates back to the 1970s, with LC-MS
profiling beginning in the 1990s [6]. MS/MS spectral tagbased annotation for non-targeted profiling, in which all of
the metabolite peaks are registered without bias but a
number of peaks remain unidentified in terms of the
chemical structure they represent, is currently extensively
used in the global characterization of plant metabolomes
[31]. The choice of analytical tools depends on the purpose
of metabolite analysis, the sampling method, and the
amount and type of cells in question. In the following
sections we provide a comprehensive description of recently invented and re-discovered metabolomics technologies
(Box 2), and their applications (Table 1).

Box 2. Analytical technologies and terms related to single cells and single cell types in plants
Metabolite feature
Metabolite features are defined as molecular entities or ion types with
a unique mass-to- charge ratio (m/z) and retention time. Features
usually arise from metabolites and their isotopes, salts, multimers,
isomers, adducts, and multiple charged ions. After being successfully
annotated with chemical characteristics, the features are known as
metabolites. Each identified metabolite can be represented by multiple features across a mass spectrum.
GC
Gas chromatography (GC) uses an inert gas such as helium as the
mobile phase and a microscopic layer of polymer (e.g., polyethylene
glycol) on a solid support as the stationary phase. Gaseous compounds
are separated by interacting with the stationary phase inside a glass or
metal tube (column). GC is used for separating volatile compounds or
compounds that become volatile on derivatization.
LC
Liquid chromatography (LC) uses a mixture of solvents (e.g., water
and methanol) as the mobile phase and various granular materials
made of solid particles (e.g., silica and polymers) as the stationary
phase. Compounds are separated based on their interaction with the
mobile phase and stationary phase. There are many different types of
LC, which can separate many different classes of compounds.
UPLC
Ultra performance liquid chromatography (UPLC) employs small
particle (1.7 mm) stationary phase beads to enhance chromatographic
resolution over a wide range of flow rates, thus improving the
efficiency, speed, and reproducibility of the separation. UPLC requires
only small sample amounts because of increased peak intensities
with reduced chromatographic dispersion.
MS
Mass spectrometry (MS) is an analytical technique that is used to
measure mass per unit charge, which reflects the elemental or
isotopic signatures of molecules. A mass spectrometer consists of an
ionization source, a mass analyzer separating ions according to the
mass-to-charge ratio (m/z), and a detector to register the ions [or
Fourier transform (FT) of image current]. The ionization methods
include electron ionization (EI), chemical ionization (CI), electrospray

640

ionization (ESI), atmospheric pressure chemical ionization (APCI) and


matrix-assisted laser desorption/ionization (MALDI). The mass analyzers include time of flight (ToF), quadrupole (Q), ion trap, linear ion
trap (LIT), and FT ion cyclotron (FT-ICR) [30].
MS/MS
Tandem mass spectrometry (MS/MS) employs two stages of MS to
examine selectively the fragmentation of a particular molecular ion.
The fragments (product or daughter ions) are used to elucidate the
molecular structure of the precursor (or parent) ion of, for example, a
metabolite or peptide.
GC-(EI)-MS
GC-MS is usually performed with EI, and sometimes with CI. For ion
separation, single quadrupole (Q) instruments are popular, but ion
traps, ToF and tandem MS are also available.
LC-MS
LC may be coupled to a variety of mass spectrometers through
atmospheric pressure ionization (API). API consists of at least three
types of ionization techniques: ESI, APCI, and photoionization, of
which ESI is the most common.
CE-MS
Capillary electrophoresis-MS (CE-MS) is a tool for the detection of
specific polar (ionic) groups of metabolites (often enriched in central
metabolic pathways), such as amino acids, organic acids, nucleotides,
and sugar phosphates [88]. Owing to low stability and technical
challenges, CE-MS for plant single-cell metabolomics has found
limited application and its wider application is daunting [89].
Recently, using patch clamp electrophysiological recording for
whole-cell visualization alongside a patch pipet to withdraw 3
picoliters of cytoplasm for metabolomic analysis by CE-MS allowed
the identification of 60 metabolites (mostly polyamines, amino
acids, and nucleotides). This advancement resulted in distinguishing
the chemical heterogeneity between GABAergic and non-GABAergic
neurons in sliced brain samples [90].
ToF
ToF mass spectrometers offer higher mass resolution than quadrupole
and ion traps, but suffer from reduced dynamic range. ToF instruments

Feature Review
are often used in untargeted metabolite profiling because the resultant
high-resolution spectra facilitate identification of unique peaks.
MALDI-ToF-MS
MALDI is a soft ionization technique that maintains molecular
structures of molecules. MALDI-ToF-MS has found several applications in single cell metabolomics [35,40,79]. Although MALDI-MS can
be applied to single cells, only a few metabolites can be identified.
MALDI-ToF-MS is known to be accurate and quantitative for
metabolite profiling at the single-cell level [91].
ESI
ESI is an ionization technique that operates at atmospheric pressure
and maintains molecular structures of molecules to yield both
positive and negative ion spectra.
Nano-ESI
Nanoelectrospray-ESI has broad applications because it utilizes spray
needles made from glass capillaries with fine tips to allow nanoliter
flow induced by ESI. It has low ion suppression and high sensitivity of
detection.
MAMS
Microarrays for mass spectrometry (MAMS), introduced in 2010, is an
analytical platform for high-throughput analysis of single cells that
involves robotic aliquoting of cell contents or suspensions followed
by MS and spectroscopic approaches: for example, nano-ESI-MS of
micropipette sampled single cell content, MALDI-MS of microfluidic
chip-assisted sample spots, imaging by MALDI-MS [20], or Fourier
transform-infrared (FT-IR) spectroscopy.
FT-ICR-MS
Fourier transform ion cyclotron MS (FT-ICR-MS) allows accurate
measurement of ions based on cyclotron frequency of the ions in a
high magnetic field. It provides super-high-resolution analysis that
can be used to determine masses with high accuracy. However, its
high cost and demanding maintenance have limited its widespread
use in metabolomics.

Single-cell metabolomics applications


Single-cell metabolomics is of paramount interest when
one considers that the sum of individual cells function and
interaction translates to function at the tissue, organ, and
organismal levels. The difficulty in obtaining homogeneous
populations of a given single cell type is the primary
driving force for the advancement of technologies for studies on individual cells (Table 1, Figure 1). However, a major
constraint to single-cell-type analysis is the extremely
limited amount of proteins and metabolites available in
a single cell (e.g., at nanomolar concentration or lower
[32]). One of the foremost developments in the area of
single-cell metabolomics consists of novel microsampling
techniques integrated with matrix-assisted laser desorption/ionization (MALDI)-MS and electrospray ionization
(ESI)-MS analysis. For example, laser microsampling of
anthocyanins in single petal cells of wishbone flowers
(Torenia spp.) was reported in 2006 [33]. Using laser
ablation ESI-MS, analysis of individual cells of onion
(Allium cepa) and daffodil (Narcissus pseudonarcissus)
bulb epidermis allowed the identification of 35 metabolites,
including amino acids, oligosaccharides, organic acids, and
alkaloids [34]. More recently, the combined application of a
cell pressure probe with UV-MALDI-time of flight (ToF)MS allowed in situ picoliter-scale sampling and metabolite
profiling of sugars, organic acids, amino acids, and specialized metabolites in single leaves and bulb cells of tulip
(Tulipa suaveolens) [35]. The pressure probe facilitated the

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Q-LIT
A quadrupole-linear ion trap (Q-LIT) hybrid instrument provides triple
quadrupole (QqQ) scans as well as high-resolution LIT scans. Q-LIT is
of high efficiency because of the short time needed to fill the trap and
the ability to yield high-quality LIT spectra. It is capable of switching
rapidly between QqQ and LIT modes to acquire LIT MS/MS
confirmatory data on an LC timescale.
QToF
Quadrupole time of flight (QToF) hybrid mass spectrometers provide
highly reliable information because of the combination of LC
retention time, accurate precursor mass, and high-quality collisioninduced mass spectra obtained by the ToF analyzer. However, the
sensitivity and duty cycle of QToF instruments are lower than for QqQ
instruments, which limits their application in quantification.
MRM
Multiple reaction monitoring (MRM) allows specific isolation of a
precursor ion for collision-induced fragmentation (CID) and scanning
of a specific product ion following fragmentation. MRM-MS is known
for its high sensitivity and selectivity in quantitative analysis, and
MRM in combination with LC has been used as the gold standard for
identification and quantitative analysis of small molecules.
NMR
Nuclear magnetic resonance (NMR) spectroscopy is a valuable
analytical platform for single-cell metabolite analysis owing to its
inherent quantitative nature and the wealth of chemical information it
can provide, but its low sensitivity, low throughput, and requirement
for large sample amounts limit its applications [44,54].
Cryogenic NMR
Cryogenic probe technology uses an ultra-cold probe, which shows
reduced noise levels. The low sensitivity of natural abundance NMR
spectroscopy is sufficiently compensated by this cryogenic probe
technology, thus enabling analysis with low amounts of samples (at
nanogram level).

fully controlled sampling of cells situated at different


depths in the tissues, quantification of sample volume,
and the addition of internal standards. Furthermore, direct single-cell analysis by nano-ESI tip aspiration has
been developed to measure metabolites in their native
environment. Aspiration of wilde malva (Pelargonium
zonale) leaf stalk single-mesophyll-cell content with a
nano-ESI tip followed by MS revealed over 1,000 features
from a sample of 1 to 5 picoliters. MS and MS/MS allowed
the identification of a-terpineol, geraniol, geranic acid, and
caffeine [36]. Similarly, a nano-ESI tip was used to extract
cellular contents from single cells of leaf, stem, and petal in
wilde malva. Then nano-ESI-MS enabled the identification
of terpenoid hydrocarbons and isoprenoids among several
other compounds that were reported for the first time in
this species [37]. These examples illustrate the accelerating efforts in single-cell metabolomics to address the longstanding challenge of identifying the minuscule quantities
of metabolites present in individual cells.
Recent technological advances that allow metabolic
profiling of isolated single cells include microarrays for
MS (MAMS) and MS imaging (MSI) [19,38]. MAMS targets
have an array of hydrophilic spots in an omniphobic environment that allows for aliquoting of picoliter- to nanolitersized droplets typically containing 04 cells. With MAMS,
two complementary platforms, (i) microspectroscopic mapping using Raman and fluorescence imaging, and (ii)
MALDI-MSI, were used sequentially to analyze the
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Feature Review
encystment of freshwater algae (Haematococcus pluvialis)
by monitoring astaxanthin, b-carotene, chlorophylls, ATP,
and ADP in the cells. Additionally, the reduction in ATP/
ADP ratio and the accumulation of astaxanthin were
shown to be correlated [39]. In another study, a MAMS
platform coupled to vacuum MALDI-linear ion trap (LIT)
was applied for imaging of epicuticular lipids on the surface of the model plant Arabidopsis (Arabidopsis thaliana)
leaves and roots. This method facilitated direct MS identification of lipids at single-cell resolution [40].
MSI uses mass spectrometers (instead of microscopes)
to detect the spatial distribution of molecules including
metabolites (instead of microscopic structures) by their
mass-to-charge ratios. The aforementioned sampling technologies (Box 1) can be combined with MSI for single-cell
metabolomics. For example, a combination of LMD and
matrix-free laser desorption/ionization (LDI)-ToF-MS
allowed localization of specialized metabolites at a resolution of 10 mm in St. Johns wort (Hypericum perforatum),
Hypericum reflexum and Arabidopsis [41]. The study
revealed that naphthodianthrones such as hypericin and
pseudohypericin are localized to the dark glands on the
leaves, and to ovary walls, stamens and stigmatic papillae,
whereas biflavonoids are found in pollen grains. In addition, flavonoids such as kaempferol, quercetin and isorhamnetin and their glycosides are present in petal and
sepal cells. Clearly, MSI allows in situ detection of metabolites and bypasses metabolite extraction steps. However,
it only allows the study of abundant metabolites (often in
the millimolar range), such as sugars and central carbon
metabolites, and those amenable to imaging (e.g., anthocyanin pigments), and therefore it is unable to provide
information on certain metabolites that are known to be of
interest: for example, phytohormones, which are typically
found at below micromolar concentrations in single cells
[22]. It is interesting to note that micro-imaging based on
NMR has been used as a non-invasive technique for plant
metabolite analyses in a few instances [4244]. Microimaging NMR takes advantage of significant water contents in several organelles, the cytoplasm, and the apoplast
for 1H-NMR spectroscopy, which provides relatively high
sensitivity and high-resolution (525 microns) measurement of the distribution of biomolecules based on magnetic
resonance frequency. This method has allowed, for instance, non-invasive mapping of abundant primary metabolites such as sucrose and amino acids in xylem and phloem
parenchyma cells of castor bean (Ricinus communis) seedlings [44].
It is noteworthy that even subcellular metabolomics has
become practicable (Table 1). For example, GC-MS and
Fourier transform (FT)-MS analyses of barley vacuoles
isolated by silicon oil centrifugation allowed the detection
of 59 primary and 200 specialized metabolites [45]. Given
that vacuoles are one of the largest organelles, and among
the easiest to isolate, the application of metabolomics to
most other subcellular compartments remains challenging. Progress in subcellular chemical phenotyping will
benefit from the invention of versatile, non-destructive,
high-resolution, and quantitative technologies. At present,
single-cell metabolomics is still largely at the stage of
technical development and metabolome cataloging.
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Single-cell-type metabolomics applications


Although single cell populations are not necessarily homogeneous (with differences in developmental and/or physiological states), they dominate the current literature (Table
1) because of the limited sensitivity of individual cell
analysis. Single-cell-type metabolomics can provide substantial improvement in understanding cellular regulatory
and metabolic processes over analyses at the tissue or
organ level. In fact, it has been stated that RNA obtained
from single cell types could be more suitable for the discovery of gene regulatory networks than RNA isolated from
whole organs because the former provides cell-type-specific
information that is missed in tissue or organ analysis [11].
Thus, the ability to separate cell types provides expanded
scope for the understanding of cell physiology, behavior,
development, and interaction with the environment. For
example, several omics approaches, including metabolomics in the mesophyll [2326] and guard-cell [23,24,4649]
systems, have revealed interesting insights into the physiology and metabolism of leaf cell types.
In the past decade, considerable progress has been made
towards understanding the metabolomes of single cell
types from numerous plant species. Trichomes have been
extensively assayed, and studies have been conducted on
sieve tube (phloem) exudates, root hair, pollen, fiber cells,
fruit epithelial cells, and guard cells (Table 1, Figure 1),
Furthermore, LMD-based time-intensive sampling has
been performed on several difficult cell types (such as
synergids, egg cells, and central cells, which are rare,
and cambial initials or companion cells, which are difficult
to access). Here we discuss metabolomics advances relating to different cell types.
Secretory cavities and epidermome
Secretory cavities/glands that are subprotodermal in origin have been reported in many families of vascular plants;
however, our knowledge of the chemical components and
functions of the structures is limited. Integration of cryogenic 1H NMR spectroscopy and high performance liquid
chromatography (HPLC) analysis of samples prepared by
LMD allowed the localization of methoxyphenylphenalenones to the secretory cavities of Dilatris pillansii, Dilatris
viscosa, and Dilatris corymbosa leaves and flowers [50].
Using GC and GC-MS, several mono- and sesquiterpenoids
were identified from the subdermal secretory cavities of
leaves of several Eucalyptus species [51]. Madagascar rosy
periwinkle (Catharanthus roseus) is the commercial source
of the anticancer drug vinblastine. The epidermome, representing the secretome (i.e., secreted metabolome) of the
epidermis from leaves of this species, was characterized
using LC-ESI-MS, leading to the identification of oleananetype triterpenoids [52]. These studies illustrate the utility
of current metabolomics technologies in analyzing natural
products of specific plant-cell populations.
Phloem parenchyma and stone cells
Bark phloem parenchyma cells and stone cells (sclereids) of
Norway spruce (Picea abies) are implicated in defense
against bark beetles [53]. Using LMD sampling of the
two cell types and cryogenic 1H NMR, several phenolics,
a stilbene glucoside, and catechin were localized to these

Feature Review
phloem parenchyma cells [43], whereas the stone cell
metabolite analysis detected two major phenolic compounds, astringin (a stilbene), and dihydroxyquercetin (a
dihydroflavonol) [54]. In pumpkin (Cucurbita maxima),
phloem exudates from cut sieve tubes of the extrafascicular
phloem yielded 80 metabolites of central metabolism using
GC-ToF-MS, HPLC, and flow injection electrospray (FIE)MS approaches [55]. The results showed metabolomic
responses after wounding, highlighting the functions of
sieve tubes in defense against herbivores. Thus, LMD
coupled to 1H NMR was successfully applied to semi-targeted profiling of phenylpropanoids in Norway spruce,
whereas MS platforms provided more global profiling of
pumpkin, indicating that single-cell-type metabolomics
can provide case-specific information on metabolomes.
Root hairs
Soybean (Glycine max) root hairs are sites for nodulation
by the nitrogen-fixing symbiotic bacterium Bradyrhizobium japonicum. A metabolomic study was performed to
identify root hair metabolites that are potentially involved
in the rhizobial infection process. Root hairs were stripped
from roots by gentle stirring of roots in liquid nitrogen. GCMS and UPLC-QToF-MS analyses detected a total of 2,610
small molecular features in soybean root hairs and
stripped roots, but only 600 were identifiable as assigned
metabolites. Of the identified compounds, 200 and 350
were identified using GC-MS and UPLC-MS, respectively,
and 166 metabolites were responsive to the symbiotic
bacterium [56].
Pollen and fiber cells
Pollen germination and tube growth are central to angiosperm reproduction. To obtain a global view of metabolic
processes in pollen, over 250 metabolites were detected in
the pollen of Easter lily (Lilium longiflorum) using a nontargeted GC-MS approach [57]. The data showed the metabolic status of germinating pollen, in which glycolysis,
tricarboxylic acid (TCA) cycle, glyoxylate cycle, starch and
fatty acid degradation were activated to provide energy
during germination and tube growth.
Like pollen tubes, cotton (Gossypium hirsutum L.) fiber
cells located on the seed epidermis elongate via tip growth
[58]. GC-MS-based metabolite profiling of 37 non-polar and
59 polar metabolites in combination with transcriptomic
analyses indicated that signaling and metabolic pathways
are coordinated to promote cell elongation in the early
stage and to support cellulose synthesis in the later stage
of cotton fiber development. Seven metabolic pathways
involving several specialized metabolites, fatty acid, and
carbohydrate metabolites displayed marked changes during cotton fiber development [59]. Another study identified
an unexpected metabolite, g-aminobutyric acid (GABA),
and thereby led to the proposed functioning of a GABA
shunt during cotton fiber development. Coincidently,
GABA, 2-ketoglutarate, succinate, and malate were found
to be higher in fibers of a cotton mutant with a reducedfiber-cell-length phenotype [60]. Higher levels of TCA organic acids present in the mutant fiber indicated higher
nitrate assimilation and GABAs possible involvement in
the regulation of nitrogen metabolism and cotton fiber

Trends in Plant Science October 2014, Vol. 19, No. 10

elongation. Thus, studies on fiber cells and pollen have


not only revealed the metabolic status of the cells in terms
of energy production but also identified key metabolites,
such as GABA, that are involved in the regulation of
cellular growth and metabolism.
Fruit epithelial and parenchyma cells
Citrus species are among the most important fruit crops
grown worldwide. Essential oils in the citrus peel have
been processed for flavor, fragrance, aromatherapy, and
industrial cleaning solvents [61]. Epithelial cells in the
citrus peel have been proposed as the site of synthesis of
essential oils. To evaluate this hypothesis, grapefruit (Citrus paradisi) epithelial cells and parenchyma cells (as
controls not producing essential oils) were collected by
LMD and pressure catapulting from the peel, followed
by metabolomics employing GC, GC-MS, and UPLC-QToF
MS [62]. The results showed the production of essential oil
metabolites, including monoterpenes, prenylated coumarins, and polymethoxylated flavonoids, in the epidermal but
not parenchyma cells. The metabolic data are further
supported by the expression patterns of genes involved
in essential-oil biosynthesis in the epithelial cells, demonstrating an important metabolic specialization of this cell
type and a chemical dimorphism of cell types with regard to
terpenoids and flavonoids. In this case, metabolomics in
coherence with transcriptomics provided insights into the
metabolomic dichotomy of these two distinct cell types
engaged in differential biological functions.
Guard cells
Guard cells form stomata on aerial surfaces, and stomatal
movement regulates transpiration, CO2 intake, and pathogen infection. Recently, guard cell protoplasts from Arabidopsis (wild type) and a heterotrimeric G-protein a
subunit null mutant, gpa1 (with ABA-hyposensitive stomata), were evaluated using targeted metabolomics. ABAresponsive metabolic changes in 85 signaling-related metabolites in guard cells were identified and quantified
using an LC-multiple reaction monitoring-(MRM)-MS
method. The metabolic changes were grouped together
in temporal modules [23]. The targeted MRM-based profiles of the Arabidopsis guard cell metabolomes provide the
first example in plants of metabolomic time course profiling obtained at single-cell-type resolution. The study
showed that 41 metabolites in wild type guard cells showed
significantly different time courses after ABA treatment,
whereas only 16 metabolites were altered by ABA in gpa1
mutant guard cells. In addition, many of the changes in
guard cells were not observed in mesophyll cells, indicating
that guard cells possess a specialized ABA responsive
metabolome. In particular, the study revealed that ABA
rapidly affects the concentrations of other hormones in a
guard-cell-specific manner; for example, indole-3-acetic
acid (IAA) levels were decreased by ABA in wild type guard
cells but not in gpa1 guard cells. The results support a new
model in which ABA operates upstream of other hormones
to regulate important processes underlying stomatal
movement. Furthermore, this study corroborated transcriptomics studies that provided evidence for cross-talk
at the transcriptional level between phytohormones:
643

Feature Review
that is, ABA and methyl jasmonate [47], and IAA [23].
Thus, alongside other studies, global metabolomics
of this specialized cell type has shown utility in further
understanding the hormonal crosstalk and regulation
of stomatal movement that is important for plant
growth, development, yield, and response to environmental factors.
Trichomes
Trichomes are metabolic hotspots for biosynthesis, regulation, and release of numerous volatile phytochemicals used
by plants for interacting with the biotic environment.
Many of the chemicals synthesized in trichomes are also
used by humans as flavorants, perfumes, and pharmaceuticals. Using microcapillaries, a total of 200 individual
Arabidopsis epidermal cells were sampled from pavement,
basal, and trichome cell types for GC-ToF-MS analysis.
The study led to identification and quantification of 117
primary metabolites, of which 38 exhibited differential
localization by cell type [63]. Furthermore, 13 glucosinolates were quantified in Arabidopsis glandular trichomes
using UPLC-ESI-QToF-MS [64].
Metabolomes of trichomes were extensively profiled by
LC-MS in several species of Solanum, enabling the identification of 119 metabolites, including terpenoids, flavonoids, fatty acids, alkaloids, and acyl sugars [65].
Furthermore, comparative transcriptomics studies corroborated the findings from metabolomics, and suggested that
photosynthetic carbon fixation contributed to specialized
metabolite biosynthesis. In glandular trichomes of the wild
tomato species Lycopersicon hirsutum, GC-MS based profiling indicated that these cells are the major sites of
methylketone storage [66]. To characterize the metabolic
diversity of glandular trichomes in cultivated and wild
tomato, a study generated chromosomal introgression
lines and followed a two-pronged approach: GC-MS to
measure terpenes in parallel with LC-ToF-MS to analyze
non-volatile specialized metabolites. Results indicated genotypic differences in the accumulation of terpenes, acylsugars, and acylsugar metabolites across several
introgression lines [67]. In another species, sweet wormwood (Artemisia annua L.), which is the source of the
antimalarial drug artemisinin, GC-MS-based profiling of
glandular trichomes indicated a similarity with the chemical constituents of entire leaves, including the presence of
artemisinic alcohol, artemisinic aldehyde, dihydroartemisinic alcohol, and dihydroartemisinic aldehyde [68]. In
addition, experiments using cannabis (Cannabis sativa
L.) trichomes allowed the identification and quantification
of nine cannabinoids by LC-MS and NMR [69]. In the
peltate glandular trichomes of Himalayan mint (Colquhounia coccinea var. mollis), the sesquiterpenoid colquhounoids A, B, and C, 15-carbon defense-related compounds,
were identified following LMD sampling of the glands and
UPLC-MS/MS and X-ray diffraction analysis of metabolites [41]. In the trichomes of coyote tobacco (Nicotiana
attenuate), nicotine, phaseoloidin, fatty acids, and acylsugars were identified using 1H NMR and LC-QToF-MS [70].
The peltate glands of basil (Ocimum basilicum) were found
to be the sites of synthesis and storage of phenylpropenes
(eugenol, chavicol, and their derivatives) using GC-MS and
644

Trends in Plant Science October 2014, Vol. 19, No. 10

HPLC, and hence are an ideal cell type for studying


phenylpropene biosynthesis [71]. Clearly, trichomes have
been used to gain substantial knowledge regarding the
biosynthesis and accumulation patterns of specialized metabolites that have roles in plant defense and are pharmaceutically important.
Compared to the isolated single cell types described
above, there are numerous metabolomics studies using
suspension culture cells [72] and single-celled algae [73].
Because of space limitations, we only briefly mention these
two cell types. The major advantages of suspension cell
cultures are the ease of propagation and handling, robustness of protocols, high homogeneity, availability of nonlimiting amounts of cell mass, and relatively stable preparations. Conversely, cultured cells are dedifferentiated,
and their metabolites could be different from those of the
original cell types, hence the results obtained may not be
directly translatable to differentiated plant cells in situ.
Another single cell type is the natural algal cell. Most of the
metabolomics studies on algae have focused on specialized
metabolites with commercial importance in food or pharmaceuticals, or have involved metabolite analyses aimed
at understanding the effects of various stresses on algae
[73].
Concluding remarks and future prospects
Metabolomics at the single-cell and single-cell-type level
remains technically challenging owing to several intrinsic
limitations, including small sampling volumes, low quantities of metabolites, and inadequate sensitivity of analytical
instrumentation [28,32]. In single-cell studies, choosing a
representative single cell is difficult because cell-to-cell
variation of metabolism can be huge. Handling, treatment,
and data recording may introduce stochastic processes into
the reported cellular metabolism. Another bottleneck is the
lack of sufficiently sensitive analytical tools [32]. Although
recent examples from single animal cells (e.g., neurons)
indicate that the CE-ESI-ToF-MS approach is able to detect
more than 300 metabolites [74], similar-scale endeavors and
deep biological insights in plant single-cell analyses are still
awaited. Single-cell analyses using lab-on-a-chip (LOC)
devices have been carried out in mammalian and microbial
cells [75]. The LOC microfluidic single-cell cultivation and
lysis methods, and the integration with flow cytometry and
advanced MS have redefined the role of single-cell analyses
in spatial and temporal resolution [76]. Analogous technologies, upon application to plant systems, will aid in holistic
and quantitative monitoring of plant cellular metabolic
dynamics: for example, as a function of genotypic and phenotypic differences. By contrast, a few studies on populations of single cell types have now shown the feasibility of
high-resolution, in-depth analysis of plant metabolomes.
Notable gray areas in single-cell and single-cell-type
metabolomics are the need for more single-cell-type model
systems, the need for more efficient extraction of metabolomes from limited biomass to improve absolute quantification, and the need for the development of analytical
platforms to enhance sensitive and quantitative measurements. Given that stochasticity in gene expression and
protein-level measurement is exhibited at the spatio-temporal dimension of single cells [77], stochasticity is naturally

Feature Review
associated with single-cell metabolomes as well. Owing to
the lack of synchronization of each individual cell in a
population and the different developmental stages and
functions with which they can be associated, stochasticity
is difficult to overcome in single-cell metabolomes. For
better or worse, the heterogeneity arising from stochasticity
can be averaged out in single-cell-type studies. Interpretation of metabolomics data [78] is stymied by poor annotation,
sheer structural diversity of metabolites, and a large number of features and unknowns contained in the data sets [28].
Further challenges lie ahead in the integration of genomics,
transcriptomics, proteomics, and metabolomics data before
we can gain a holistic understanding of cellular physiology
and development. Nevertheless, current advances indicate
a potential paradigm shift from tissue/organ-scale metabolomic studies to single-cell metabolomics, which will complement other omics methods on the way towards a unified
systems biology of single cells.
Acknowledgments
This work was supported by the US National Science Foundation grants
NSF-MCB-0817954, NSF-MCB-1157921, and NSF-IOS 1025837 to S.M.A.
and NSF grants NSF-MCB-0818051, NSF-MCB-1158000, and NSFCAREER-0845162 to S.C. We thank Mengmeng Zhu for helpful comments
on the manuscript. We apologize to all the colleagues whose work could not
be cited owing to space constraints. The authors appreciate the comments
of three anonymous reviewers that improved this article.

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