The Plant Journal (2021) doi: 10.1111/tpj.

15261

TECHNICAL ADVANCE

Mass spectrometry-based quantification and spatial

localization of small organic acid exudates in plant roots
under phosphorus deficiency and aluminum toxicity
David Gomez-Zepeda1,‡,†, Moises Frausto1,2,†, He
ctor-Rogelio Na
jera-Gonza
lez1,§,†, Luis Herrera-Estrella1,2,* and Jose
-Juan
Ordaz-Ortiz1
1
Unidad de Geno
mica Avanzada, Centro de Investigacio
n y de Estudios Avanzados del Instituto Polite
cnico Nacional, Irapu-
ato 36824, Mexico,
2
Institute of Genomics for Crop Abiotic Stress Tolerance, Department of Plant and Soil Science, Texas Tech University, Lub-
bock, TX 79409, USA

Received 16 November 2020; accepted 26 March 2021.

*For correspondence (e-mail lherrerae@cinvestav.mx; jose.ordaz.ortiz@cinvestav.mx).
†
These authors made equal contributions to this work and are considered joint first authors.
‡
Present address: University Medical Center of the Johannes Gutenberg-University Mainz, Institute for Immunology, Mainz, 55131, Germany
§
Present address: Innovak Global, Chihuahua, 31375, Mexico

SUMMARY
Low-molecular-weight organic acid (OA) extrusion by plant roots is critical for plant nutrition, tolerance to
cations toxicity, and plant–microbe interactions. Therefore, methodologies for the rapid and precise quantifi-
cation of OAs are necessary to be incorporated in the analysis of roots and their exudates. The spatial loca-
tion of root exudates is also important to understand the molecular mechanisms directing OA production
and release into the rhizosphere. Here, we report the development of two complementary methodologies
for OA determination, which were employed to evaluate the effect of inorganic ortho-phosphate (Pi) defi-
ciency and aluminum toxicity on OA excretion by Arabidopsis roots. OA exudation by roots is considered a
core response to different types of abiotic stress and for the interaction of roots with soil microbes, and for
decades has been a target trait to produce plant varieties with increased capacities of Pi uptake and Al toler-
ance. Using targeted ultra-performance liquid chromatography coupled with high-resolution tandem mass
spectrometry (UPLC-HRMS/MS), we achieved the quantification of six OAs in root exudates at sub-micro-
molar detection limits with an analysis time of less than 5 min per sample. We also employed targeted
(MS/MS) matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) to detect
the spatial location of citric and malic acid with high specificity in roots and exudates. Using these methods,
we studied OA exudation in response to Al toxicity and Pi deficiency in Arabidopsis seedlings overexpress-
ing genes involved in OA excretion. Finally, we show the transferability of the MALDI-MSI method by ana-
lyzing OA excretion in Marchantia polymorpha gemmalings subjected to Pi deficiency.

Keywords: aluminum toxicity, Arabidopsis thaliana, high resolution mass spectrometry, Marchantia poly-
morpha, mass spectrometry, mass spectrometry imaging, organic acids, phosphorus deficiency, root exu-
dates, technical advance.

INTRODUCTION
Low-molecular-weight organic acids (OAs) are major com-
ponents of root exudates, not only because of their high
abundance relative to other compounds present in root
exudates, but also because of their crucial roles in plant
nutrition (Mimmo et al., 2014; Ryan et al., 2001), heavy
metal solubilization and detoxification (Huang et al., 1998;
Kochian et al., 2015; Montiel-Rozas et al., 2016), pH modifi-
cation of the rhizosphere (Guo et al., 2010), and responses
to hypoxia (Choi and Roberts, 2007) and as bio-stimulants
and chemoattractants (Macias-Benitez et al., 2020; Zhang
et al., 2014), as well as their contribution to the carbon

© 2021 Society for Experimental Biology and John Wiley & Sons Ltd 1
2 David Gomez-Zepeda et al.
cycle and biological weathering (Adeleke et al., 2017). In
agriculture, inorganic ortho-phosphate (Pi) deficiency and
aluminum (Al) toxicity are the two main constraints for
crop productivity in acidic soils, forming part of what is
known as acid soil syndrome (Kochian et al., 2004). Over
half of the potentially arable lands are Pi-deficient or acidic
or contain toxic levels of Al3+ that drastically inhibit root
growth (Kochian et al., 2015; Lynch, 2011; von Uexku € ll &
Mutert, 1995). The exudation of OAs by plant roots to the
rhizosphere is an effective strategy to cope with cation tox-
icity and, in some cases, Pi deficiency (Delhaize et al.,
2009; Kochian et al., 2004; Teodoro et al., 2019; Veneklaas
et al., 2003). OA-dependent Pi mobilization is given mainly
by ligand exchange, whereas toxic cations such as Al3+ are
excluded from the root tip or chelated as non-toxic OA–
metal complexes (Gerke et al., 1994; Kochian et al., 2015;
Kopittke et al., 2017).
Engineered plants with enhanced OA exudation in roots
have been assessed for more than two decades as a strat-
egy to improve growth under Pi deficiency and Al toxicity,
mainly by overexpressing genes involved in the synthesis
or transport of OAs. The malate transporter ALUMINUM-
ACTIVATED MALATE TRANSPORTER 1 (ALMT1) and its
positive transcriptional regulator SENSITIVE TO PROTON
RHIZOTOXICITY 1 (STOP1) are two major components in
Arabidopsis that play an important role in tolerance to Al
toxicity (Delhaize et al., 2004; Hoekenga et al., 2006; Iuchi
et al., 2007) and the modification of root architecture in
response to Pi deficiency (Balzergue et al., 2017; Mora-
Mac
ıas et al., 2017). In addition to ALMT1, STOP1 posi-
tively regulates the transcription of MULTI-DRUG AND
TOXIC COMPOUND EXTRUSION (MATE), a citrate trans-
porter-encoding gene (Liu et al., 2009). It has been consis-
tently reported that plant varieties with increased OA
exudation rates have enhanced Al3+ tolerance (de la
Fuente, 1997; Delhaize et al., 2004; Magalhaes et al., 2007;
Tesfaye et al., 2001). However, the reports are often con-
tradictory regarding the increase of P uptake efficiency,
especially in non-acidic pH (Delhaize et al., 2001; Leiser
et al., 2014; Lu€ et al., 2012; Neumann and Ro € mheld, 2007;
Silva et al., 2018). Methodologies for the rapid and precise
quantification and localization of OAs in root exudates are
necessary to better understand their role in the response
to P deficiency and to evaluate engineered plants. More-
over, because members of the ALMT family often present
significant permeability for more than one anion (Gruber
et al., 2010, 2011; Kovermann et al., 2007; Meyer et al.,
2011; Pineros et al., 2008; Sasaki et al., 2004), it is neces-
sary to expand the profile of OAs assessed in root exu-
date analysis.
Diverse methods have been employed for the quantifica-
tion of OAs in root exudates; for example, reversed-phase
(RP) high-performance liquid chromatography (HPLC) (Lyu
et al., 2016) and capillary electrophoresis (Hoekenga et al.,
© 2021 Society for Experimental Biology and John Wiley & Sons Ltd,
2003; Pin ~ eros et al., 2002) coupled to UV absorbance detec-
tion (at 214 or 232 nm) have been widely used. However,
UV detection has poor selectivity and a high detection
threshold (lowest limit of quantification [LLOQ] around
50 µM) (Jaitz et al., 2011). This limits the use of these meth-
ods to quantify OAs in root exudates whose concentrations
range between low micromolar and nanomolar levels
(Hoekenga et al., 2003; Jaitz et al., 2011; Pin ~ eros et al.,
2002). Ultra-performance liquid chromatography (UPLC)
coupled to targeted tandem mass spectrometry (MS/MS),
in which a precursor ion is filtered and then fragmented
and the product ions are detected, achieves higher analyti-
cal performance in shorter times (5–10 min) compared to
HPLC-UV methods (around 30 min) (Erro et al., 2009). For
instance, LLOQs of around 0.1 µM (0.015–0.04 µg ml 1)
have been achieved for the quantification of OAs in root
exudates using UPLC-MS/MS (Wang et al., 2015).
Beyond the quantification of total OAs in plant exudates,
it is important to determine the regions or cells in the root
that secrete OAs into the rhizosphere, which could play an
important role in Pi exchange or tolerance to toxic cations.
It has been reported that malate exudation takes place
principally at the root tips (Delhaize et al., 2004; Kopittke
et al., 2017); however, the expression of malate trans-
porters in the whole root suggests that malate may be
secreted in other root zones in P deficiency conditions (Kle-
pikova et al., 2016). For instance, a recent study in Ara-
bidopsis showed that ALMT3 is expressed mainly in root
hairs and is responsive to P deficiency, contributing to an
increased release of malate into the apoplast and the rhizo-
sphere (Maruyama et al., 2019). The differences in OA exu-
dation along the root length have been assessed either by
analyzing the exudates of pre-excised root segments or by
incubating a specific section of the root in a small chamber
(Delhaize et al., 2004; Pen ~ aloza et al., 2002; Ryan et al.,
1995; Zhu et al., 2005). These methods need meticulous
sample preparation and could result in artifacts in OA
release due to enzymatic degradation or stress-induced
changes. Therefore, there is a need for methods that allow
the in situ localization of OA exudation zones.
Matrix-assisted laser desorption/ionization (MALDI)
mass spectrometry imaging (MSI) has been proven to be a
powerful tool for exploring the spatial distribution of small
molecules in several plant tissues (Boughton et al., 2016;
Dong et al., 2016). It can provide a spatial resolution of
10 µm or even less. However, the matrices used for
MALDI-MS can generate a lot of interference ions in the
low-mass region (<800 m/z) due to matrix self-dissociation
and the formation of clusters with the analytes (matrix
adducts), which can hinder the correct identification of
metabolites (Shroff et al., 2009). Many efforts have focused
on the development of alternative matrices generating
fewer interference ions to improve sensitivity and speci-
ficity, such as the strong bases 9-aminoacridine and 1,8-bis
The Plant Journal, (2021), doi: 10.1111/tpj.15261

(dimethyl-amino) naphthalene (DMAN), which are used to
detect negatively charged ions (Silva et al., 2016). Ye et al.
(2013) used DMAN to detect diverse metabolites in gela-
tine-embedded Medicago truncatula root nodules, includ-
ing OAs as pyruvate, lactate, maleonate, succinate,
malonate, and citrate/isocitrate, which were validated
using targeted experiments. However, an alternative strat-
egy is the use of targeted MALDI-MSI to detect previously
selected ions of the analyte, which grant the location of
one metabolite at a time in each sample with a very high
specificity and sensitivity irrespective of the matrix used
(Boughton et al., 2016).
Here, we report two complementary targeted high-reso-
lution (HR) MS/MS methods for high-throughput quantifica-
tion and spatial localization of OAs in root exudates using
UPLC-HRMS/MS and MALDI-MSI HRMS/MS, respectively.
These methods were used (i) to study the in vitro release of
OAs of Arabidopsis thaliana wild-type (WT) and transgenic
lines overexpressing ALMT1 and STOP1 under treatments
of P deficiency ( P, pH = 5.7), low pH (control, pH = 4.1),
and low pH and Al toxicity (+Al, pH = 4.1); and (ii) to evalu-
ate the spatial localization of OAs in Arabidopsis root apices
under P treatments. In addition, we demonstrate the trans-
ferability of MALDI-MSI to other model plants by analyzing
Marchantia polymorpha in +P and P conditions.
RESULTS AND DISCUSSION
UPLC-HRMS/MS quantification method development
The LC-MS quantification of OAs is challenging because of
their high hydrophilicity, which results in low retention
times in RP chromatography, and the additives used in RP
LC-UV, such as H2SO4 (pH 4.6) (Zhang et al., 2014) or
KH2PO4 (pH 2.3), are not compatible with MS detection (Lyu
et al., 2016). Therefore, other chromatographic methods
have been proposed. For instance, hydrophilic interaction
liquid chromatography (HILIC) with amide groups has been
used for the separation of OAs and other polar metabolites
(Paglia et al., 2012). However, we observed that HILIC-
Amide provided poor peak shapes and accumulation of
citrate on the column (Figure S1). Erro et al. (2009)
employed an ion exclusion column (Rezex RHM-Monosac-
charide) to study root exudates of maize (Zea mays), lupin
(Lupinus albus), and chickpea (Cicer arietinum), achieving a
good separation of diverse OAs with LLOQs from 25.8 µM
(maleate) to 1135 µM (pyruvate) using a multiple-reaction
monitoring (MRM) method in a triple-quadrupole MS
instrument. Nevertheless, the HPLC-MS analysis requires
16 min per sample, which can be a hurdle for high-through-
put analysis required to evaluate multiple plant lines in
shorter times (Delhaize et al., 2009; Kochian et al., 2004;
Teodoro et al., 2019; Veneklaas et al., 2003).
We developed a targeted UPLC-HRMS/MS method for
the specific quantification of organic acids, employing a
© 2021 Society for Experimental Biology and John Wiley & Sons Ltd,
The Plant Journal, (2021), doi: 10.1111/tpj.15261
Quantification and localization of acid exudates 3
trifunctional alkyl C18-bonded stationary phase (HSS-T3
column; Waters Corporation, Milford, MA, USA) for chro-
matographic separation (Figure 1a), which has been previ-
ously used for the quantification of OAs in root exudates
(Wang et al., 2015). The analytes were correctly resolved
(Figure S2) in a 4.7-min chromatographic method, allowing
high-throughput analysis of samples. For the HRMS/MS
method (Alelyunas et al., 2014), the Synaptâ (G1) HDMS
was operated in negative electrospray ionization (ESI)
mode. The deprotonated ions [M-H] of the OAs were
selected as precursors and fragmented with optimized col-
lision energy (CE), and, whenever possible, a fragment ion
was used for enhanced duty cycle (EDC) to improve sensi-
tivity (see Experimental Procedures section) (Alelyunas
et al., 2014) (Table S1). Calibration curves were prepared in
5% methanol, 95% 0.19 MS medium with 0.1% of formic
acid (FA) to account for matrix effects of the root exudates.
Furthermore, spiked heavy isotope-labeled standards of
malate and citrate were employed as internal standards for
calibration to improve the quantification performance of
the two most important OAs in root exudates.
The UPLC-MS/MS HRMS/MS method proved to be
highly selective, sensitive, accurate, and precise. The MS/
MS spectra of the analytes in A. thaliana exudate samples
matched the standard analytes (Figure 1b) and the m/z
error was below 40 ppm (Table S2), demonstrating the
selectivity of the analysis. The isomeric specificity of citrate
quantification was proven by the presence of the citrate-
specific fragment 87 and the absence of the isocitrate-
specific fragment 73 (Erro et al., 2009) in the A. thaliana
exudates (Figure 1b, right). Although the method could be
improved to quantify more organic acids and differentiate
their isomers, we focused hereby on the quantification of
the most important OAs for plant responses to abiotic
stress. Although the Synapt HRMS (G1) employed in this
study is equipped with traveling wave ion mobility spec-
trometry (TWIMS), its resolution is not sufficient to sepa-
rate isomers of such small molecules and the lower ion
transmission efficiency when TWIMS is used can result in
a significant sensitivity loss (Shliaha et al., 2013).
Fit to curve and sensitivity were assessed in calibration
curves (Table S3, Figure S3). Quadratic curves (1/x weight-
ing) were employed and a R2 fit above 0.99 was obtained
for all analytes. The limit of detection (LOD) was below
0.25 µM for all the analytes (1.25 pmol on column), except
lactate (0.56 µM), and the LLOQ was 0.5 µM (2.5 pmol on col-
umn) except for citrate (0.1 µM) and lactate (1 µMM). This is
equivalent to sub-micromolar concentrations in the exu-
dates, providing enough sensitivity to quantify OAs in exu-
date samples which can be at the low micromolar to
nanomolar range (Hoekenga et al., 2003; Jaitz et al., 2011;
Pin~ eros et al., 2002). Accuracy in the calibrators and quality
controls (QCs) was between 85% and 120% for all the ana-
lytes and the coefficient of variation (CV) was below 20%,
4 David Gomez-Zepeda et al.

(a)
1) Plant
2) Exudate recovery 3) Sample treatment 4) Targeted MS 5) Data treatment
samples

Skyline
• Lyophylisation
UPLC-MS/MS

• Recovery in 5% UPLC-ESI(-)
MeOH + 0.1% FA HRMS/MS
• Calibration curve
0.1 to 30 µM - -
A. thaliana -
+/- Pi 24 well plate, 2 h, - - MS/MS
MS 0.1X medium Quantification

(b) Standard (5 µM) A. thaliana exudate

113.0140
100
113.0137 (c) (d)
Citrate Heavy 100
88.0091 88.0094 127.9702
%
%

(193 > 113.019) 0 m/z 0 m/z

100 200 -0 100 200

111.0093 111.0054
Citrate 100
112.0149
100
87.0066 129.0218
87.0066
%
%

(191 > 111.012) 0 m/z 0 m/z

100 200 -0 100 200

Malate Heavy 100

119.0149
100 74.0214
119.0143
74.0207 137.0276 137.0240
%
%

(137 > 119.011)

0 m/z 0 m/z
100 200 -0 100 200

Malate 115.0072 115.0033

100 100 71.0111
(133 > 114.997) 71.0111 133.0181
%

0 m/z 0 m/z
100 200 -0 100 200
α-Ketoglutarate 101.0215 145.0216 101.0252
145.0172
(145 > 101.022) 100 100
146.9436 149.0494
%

0 m/z 0 m/z
100 200 -0 100 200
Succinate
73.0251 116.9309 73.0251 117.0187
(117 > 73.024) 100
117.9308
100
%

0 m/z 0 m/z
100 200 -0 100 200
Fumarate 116.9309 100 71.0111
115.0072
100 117.9268
(115 > 71.013) 117.9268
%

99.9264
%

0 m/z 0 m/z
100 200 -0 100 200
Lactate 89.0218 89.0218
100 100
(89 > 89.024) 88.9835
%
%

0 m/z 0 m/z
100 200 -0 100 200

Figure 1. Targeted UPLC-MS/MS quantification method development.

(a) Schema of the method.
(b) Mass spectra extracted from the analysis of a calibrator containing the standard analytes at 3 µM (left) and a root exudate sample from A. thaliana (right).
(c) Accuracy and precision (coefficient of variation [CV]) in calibration curve (Cal.) and QCs. Data points in gray correspond to individual measurements.
(d) Recovery and CV from A. thaliana root exudate pools spiked with standard analytes at 0.4 or 4 µM. Data points in gray correspond to the average recovery or
the CV, respectively, for each analyte.

indicating high accuracy and precision, respectively (Fig-
ure 1c). The recovery in samples from a pool of A. thaliana
spiked with 0.4 or 4 µM of standard analytes (Figure S4) was
between 90% and 115% and the CV was below 20% (Fig-
ure 1d), proving good recovery and precision of the method
on exudate samples without significant matrix effect.
UPLC-MS/MS quantification of OA exudation in
Arabidopsis thaliana genotypes under P deficiency and
Al toxicity
We generated transgenic lines of Arabidopsis overexpress-
ing either ALMT1 (OX.ALMT1) or STOP1 (OX.STOP1) under
control of the CaMV 35S promoter. Two lines of each
construct were selected for further experiments. Quantita-
tive reverse transcriptase-PCR (qRT-PCR) analysis of RNA
extracted from Pi-deprived seedlings (n = 3) showed that
line OX.ALMT1-1 had 23-fold (P-value < 0.01) and line
OX.ALMT1-2 105-fold (P-value < 0.05) higher transcript
levels of ALMT1 that WT. OX.STOP1-1 and OX.STOP1-2
lines presented STOP1 transcript levels 15 (P-value < 0.001)
and 4 times (P-value < 0.01) higher than WT, respectively
(Figure 2a). In the OX.STOP1-1 line, the transcript levels of
ALMT1 and MATE were 10- (P-value < 0.01) and 4-fold (P-
value < 0.05) higher than in WT, whereas in OX.STOP1-2
they remained unchanged (P-value > 0.05). These observa-
tions indicate that the increase in expression of ALMT1

© 2021 Society for Experimental Biology and John Wiley & Sons Ltd,
The Plant Journal, (2021), doi: 10.1111/tpj.15261
 Quantification and localization of acid exudates 5
(a) (b)

(c) (d)

Figure 2. Transcript levels and UPLC-MS/MS TOF-MRM quantification of organic acid efflux under Al toxicity and P deficiency from A. thaliana WT and trans-
genic lines.
(a) Transcript levels (qRT-PCR) of plants grown for 8 days in +P (500 µM of P, 0 µM of Al, pH = 5.7) and for 3 days in P. For comparison between each transgenic
line and WT, unpaired two-sided t-tests were performed (n = 3, error bars = SE); significant differences are indicated with asterisks (*P < 0.05, **P < 0.01, and
***P < 0.001).
(b) Stacked bar plots of organic acid exudation rates, showing the individual proportion of each analyte in the exudate (top) and the combined exudation rates
(bottom) from each line in control (100 µM of P, 0 µM of Al, pH = 4.1), P (10 µM of P, 0 µM of Al, pH = 5.7), and +Al conditions (100 µM of P, 700 µM of Al,
pH = 4.1). For the total exudation rates of OAs, an unpaired two-sided t-test was performed (n = 8, error bars = SE) for every single comparison between WT
and each transgenic line in the corresponding treatment. The Bonferroni correction for multiple comparisons was applied per treatment and significant differ-
ences are indicated with asterisks (*P < 0.05, **P < 0.01, and ***P < 0.001).
(c) Exudation rates of organic acids in Arabidopsis lines under P deficiency and Al toxicity. Independent unpaired two-sided t-tests were performed (n = 8, error
bars = SE) comparing the WT values with those of each transgenic line in the corresponding treatment and analyte. P-values were corrected for multiple com-
parisons by the Bonferroni correction and significant differences are indicated with asterisks (*P < 0.05, **P < 0.01, and ***P < 0.001).
(d) Linear correlation analysis between the organic acids exuded in each biological sample for plants under P, +Al, and control conditions. Analytes are indi-
cated in the diagonal, the corresponding scatterplots of the quantitative measurements below the diagonal, and the Pearson correlation coefficient R above the
diagonal, for each comparison pair. Data points corresponding to control (yellow), P (red), and +Al (blue) are color-coded as in the panel in (c). Genotypes are
represented by symbols ○ (WT), + (OX.ALMT1), and ⨯ (OX.STOP1).

© 2021 Society for Experimental Biology and John Wiley & Sons Ltd,
The Plant Journal, (2021), doi: 10.1111/tpj.15261
6 David Gomez-Zepeda et al.
and MATE transporters in the OX.STOP1 lines may depend
on exceeding a certain threshold of STOP1 transcript
levels.
To validate our OA quantification method, we evaluated
both the well-known response of OA exudation to Al toxic-
ity and the response to P deficiency in WT (Col-0) and
OX.ALMT1 and OX.STOP1 transgenic lines. Because of the
large effect size between treatments, the quantitative anal-
ysis of OA exudation was performed both with untrans-
formed (Figure 2b–d) and log10-transformed (Figure S4)
data. In either case, similar conclusions were obtained. It
has been widely documented that A. thaliana Col-0 seed-
lings respond to toxic levels of Al in the medium by
increasing the exudation of malate and citrate by the roots
(Liu et al., 2009; Sharma et al., 2016). Our analysis (n = 8)
revealed that WT plants in +Al increased their malate and
citrate exudation rates 14-fold (P-value < 0.001; Cohen’s d:
8.5) and 24-fold (P-value < 0.001; Cohen’s d: 6.5), respec-
tively, compared to control conditions (Figure 2b,c, see
also Figure S5a). Malate and citrate efflux measured in the
root exudates of Al-treated Arabidopsis seedlings in our
experimental conditions is in the same order of magnitude
to values previously reported in several studies using dif-
ferent methodologies (Hoekenga et al., 2006; Iuchi et al.,
2007; Kobayashi et al., 2007; Kobayashi et al., 2013; Liu
et al., 2009).
Under our experimental conditions, the exudation rates
of malate and citrate of P-deficient WT Arabidopsis plants
do not surpass that of the control treatment (Figure 2b,c,
see also Figure S5a). However, it has recently been
reported that P-deprived Arabidopsis plants enhance their
malate exudation fourfold compared to P sufficiency condi-
tions (Maruyama et al., 2019). This discrepancy is probably
due to the difference between the studies regarding the
type of normalization of the exudation rates (normalized
by plant here instead of fresh root weight) and the applica-
tion time of P treatment in liquid medium (7 h here
instead of 20 days). It is known that the maximum rates of
malate exudation in Al-stressed roots of Arabidopsis seed-
lings occur at 4–6 h post-treatment (Kobayashi et al., 2007).
Because in our experiments we used seedlings subjected
to relatively short Al exposure times, the seedlings were
very similar in size, thus normalization by plant gives a bet-
ter outlook of the potential to exudate OAs under a specific
type of stress. It was recently reported that low pH, which
in turn interacts with other related stresses like P deficiency
and Al and Fe toxicity in acid soils, contributes to STOP1
stabilization and ALMT1 transcriptional activation (Balzer-
gue et al., 2017; Godon et al., 2019; Ojeda-Rivera et al.,
2020). Instead, the role of ALMT1 in roots of WT plants
under P deficiency may be restricted to increase malate
excretion to the root tip as a component of the meristem-
atic exhaustion mechanism that triggers the root architec-
ture modifications that characterize this nutritional stress
© 2021 Society for Experimental Biology and John Wiley & Sons Ltd,
(Gutie
rrez-Alan
ıs et al., 2018). Indeed, seedlings of
OX.ALMT1-2 and OX.STOP1-1 lines germinated in P
treatment showed a hypersensitive response in changing
its root architecture (faster root growth inhibition in all
roots and higher lateral root density) compared to the WT
(Figure S6). Recently, it has been reported that overexpres-
sion of ALMT1 and STOP1 in Arabidopsis and the con-
comitant change in root architecture in P are associated
with higher Fe3+ accumulation in the apoplast, presumably
as Fe–malate complexes (Wang et al., 2019).
ALMT1 is the main transporter regulating the efflux of
malate in Arabidopsis roots in response to +Al (Hoekenga
et al., 2006). Thus, plants overexpressing ALMT1 present a
more vigorous malate exudation in response to Al stress
(Kobayashi et al., 2013). Accordingly, OX.ALMT1-1,
OX.ALMT1-2, and OX.STOP1-1 showed a significant
increase in malate exudation in control (fold change: 2.6–
5.1; Cohen’s d: 2.2–6.5), P (fold change: 3.0–9.1; Cohen’s
d: 2.7–7.0), and +Al (fold change: 2.4–8.1; Cohen’s d: 3.0–
5.5) conditions compared to the WT in the same treat-
ments (Figure 2c, see also Figure S5a). As expected, the
variation in malate exudation rates in all lines in P and
+Al treatments is positively associated with their ALMT1
transcript levels (Figure 2a). The fold change values for
malate exudation in P conditions in our transgenic lines
are within the range of those reported in other studies
aimed to increase root OA exudation under P deprivation
without Al stress (Koyama et al., 2000; Lo
pez-Bucio et al.,
2000; Lu € et al., 2012; Wang et al., 2013; Wang et al., 2015).
In addition to positively regulating malate exudation via
ALMT1, STOP1 also regulates citrate exudation by induc-
ing MATE transcription (Liu et al., 2009). When compared
with WT, citrate exudation rates increased significantly in
the control (fold change: 4.0; Cohen’s d: 2.3) and P (fold
change: 3.6; Cohen’s d: 4.5) treatments only in OX.STOP1-
1, which has MATE expression levels four times higher
than WT (Figure 2a–c, see also Figure S5a). However, in
+Al conditions, citrate exudation rates increased only 1.5-
fold in OX.STOP1-1 (P < 0.05; Cohen’s d: 2.3) compared to
WT, most likely due to the Al-induced expression of MATE
in the WT. Interestingly, OX.ALTM1-1 and OX.ALTM1-2
presented an increment in citrate efflux similar to that in
OX.STOP1-1 in +Al conditions (fold changes: 1.5 and 1.7;
Cohen’s d: 1.5 and 1.8; compared to WT) (Figure 2c, see
also Figure S5a). This increase in citrate exudation in the
OX.ALMT1 lines may be due to partial permeability of
ALMT1 to citrate, similar to that observed in Xenopus lae-
vis oocytes expressing ALMT1 from wheat (Triticum aes-
tivum) (Sasaki et al., 2004). In the case of the OX.ALMT1
lines in P conditions, we did not observe any significant
increase in citrate exudation when compared to WT in the
same treatment. Furthermore, the citrate level in the exu-
dates is consistently lower in P compared to control con-
ditions in all genotypes (fold change: 0.19–0.59; Cohen’s d:
The Plant Journal, (2021), doi: 10.1111/tpj.15261

1.1–2.4), which suggests that, unlike Al-stress, citrate
makes a small contribution to the OAs excreted by the
roots of P-deprived Arabidopsis.
In addition to malate and citrate, our quantification
method allowed us to evaluate a-ketoglutarate, succinate,
fumarate, and lactate. As expected, the two main compo-
nents of the total organic acids exuded from WT plants
under Al stress are malate (49%) and citrate (28%) (Fig-
ure 2b, top). Meanwhile, WT plants in control and P con-
ditions presented a more equilibrated OA profile, malate
still being the most abundant OA (28% and 40% of the
total, respectively). Interestingly, +Al treatment also
induced a significant increase in a-ketoglutarate (3.6-fold
change; P-value < 0.001; Cohen’s d: 5.7), succinate (4.2-fold
change; P-value < 0.001; Cohen’s d: 6.1), and fumarate
levels (6.6-fold change; P-value < 0.001; Cohen’s d: 3.4) in
WT exudates compared to the control treatment (Figure 2c,
see also Figure S5a). In contrast, lactate levels in exudates
remained unchanged. The presence of a high concentra-
tion of lactate in the root exudates may indicate growth
under anoxic conditions (Choi and Roberts, 2007), which
stimulates OA exudation (Ryan et al., 2001). Compared to
control treatment, P-deprived WT plants did not show any
substantial increment of any OAs evaluated. Because of
the difference in pH between the P and control treat-
ments, we cannot rule out the possibility that WT plants
responds to P deficiency by incrementing its OA exudation.
In either case, OA exudation rates in P-deficient WT plants
seem to be close to those obtained in low pH (control treat-
ment) and lower than those in plants subjected to both low
pH and Al stress.
Surprisingly, under +Al treatment OX.ALMT1-1,
OX.ALMT1-2, and OX.STOP1-1 showed significant
increases in the exudation of a-ketoglutarate (fold change:
1.3–4.8; Cohen’s d: 1.5–8.3), succinate (fold change: 3.4–
10.2; Cohen’s d: 3.3–7.8), and fumarate (fold change: 3.4–
14.3; Cohen’s d: 3.0–6.4) when compared to the WT (Fig-
ure 2c, see also Figure S5a). Similarly, there is a tendency
of increasing the exudation rates for these three metabo-
lites in the P and control conditions, although to a lesser
extent. In contrast, transgenic line OX.STOP1-2, which dis-
played ALTM1 and MATE transcript levels similar to WT,
did not present any significant increase in OA exudation in
any treatment when compared with WT levels, except for
fumarate efflux in +Al (fold change: 2.5; P-value < 0.001;
Cohen’s d: 3.6). The exudation rates of citrate, a-ketoglu-
tarate, succinate, and fumarate seem to correlate with the
levels of exudation of malate and, therefore, of ALMT1
expression. To evaluate the relationship of the exudation
values between the OAs assessed, we performed a linear
correlation analysis using all data points of each biological
replicate in all the three treatments and five genotypes.
The correlation analysis shows a strong correlation
between malate, a-ketoglutarate, succinate, and fumarate
© 2021 Society for Experimental Biology and John Wiley & Sons Ltd,
The Plant Journal, (2021), doi: 10.1111/tpj.15261
Quantification and localization of acid exudates 7
(R = 0.89–0.99), a medium correlation between citrate and
the rest of the OAs of the Krebs cycle (R = 0.71–0.79), and
a low correlation between lactate and all other OAs
( 0.097 to 0.008) (Figure 2d, see also Figure S5b). The
positive correlation between the exudation of OAs belong-
ing to the Krebs cycle is reflected in the total exudation
rates, where the overexpressing plants with higher ALMT1
transcript levels present significantly higher exudation
rates than WT plants in control (fold change: 1.7–2.7;
Cohen’s d: 1.9–3.3), P (fold change: 2.5–6.3; Cohen’s d:
2.8–6.9), and +Al (fold change: 2.3–6.7; Cohen’s d: 3.1–5.9)
conditions (Figure 2b, bottom). Moreover, the order of the
abundance of the three main OAs in the exudates changed
with respect to the WT (malate > citrate > succinate) in the
OX.ALMT1-2 and OX.STOP1-1 lines under +Al treatment
(malate > succinate > citrate) and in the OX.ALMT1-1 line
under +Al treatment (malate > succinate > fumarate >
citrate) (Figure 2b, top).
The highest levels of OA exudation in Arabidopsis are
observed under Al stress, where it is well known that
ALMT1 is activated at both transcriptional and functional
levels (Kobayashi et al., 2007). The high correlation that we
observed between the exudation of malate, a-ketoglu-
tarate, succinate, and fumarate (and to a lesser extent
citrate) suggests that ALMT1 is capable of transporting not
only malate but also other OAs, which could contribute
significantly to the Al exclusion capacity from the root tip
of Arabidopsis. Although the roles of Arabidopsis ALMT1
in malate transport under Al stress (Kochian et al., 2015),
in changes in root architecture under P deficiency
(Gutie
rrez-Alan
ıs et al., 2018), and in promoting beneficial
microbial interactions (Kobayashi et al., 2013; Rudrappa
et al., 2008) have been extensively studied, little is known
about its ability to transport other anions besides malate.
However, it is not uncommon for transporters belonging to
the ALMT family to be permeable to several OAs. For
example, Arabidopsis ALMT9 is a vacuolar malate trans-
porter expressed in mesophyll cells which is also perme-
able to fumarate, although to a lesser extent (Kovermann
et al., 2007). Similarly, Arabidopsis ALMT6 is a vacuolar
malate transporter that possesses high permeability to
fumarate and, to a lesser degree, citrate (Meyer et al.,
2011). An interesting example is transgenic ALMT1-overex-
pressing barley (Hordeum vulgare), which, similar to our
transgenic lines, presents an increased exudation of not
only malate (fold change: 20), but also succinate (fold
change: 15) and fumarate (fold change: 3), compared to
WT plants (Gruber et al., 2011). Considering all these
observations, it is possible that the ability of malate to alle-
viate Al toxicity and P deficiency in soil has been overesti-
mated in plants where the strategy was the overexpression
of an ALMT transporter and other OAs were not evaluated.
When compared with control conditions, P-deprived
OX.ALMT1 plants tend to consistently present higher
8 David Gomez-Zepeda et al.
exudation rates of malate, a-ketoglutarate, succinate, and
fumarate. These results support the idea that ALMT1 is
activated at a functional level under P deficiency in Ara-
bidopsis roots and can be used to enhance OA exudation
in an Al-independent manner in transgenic plants. Remark-
ably, transgenic plants overexpressing ALMT1 gain the
ability to respond to P conditions by exuding malate in
amounts corresponding to 37% (OX.ALMT1-1) and 61%
(OX.ALMT1-2) of that in WT plants under Al stress (Fig-
ure 2c, see also Figure S5a). In terms of total OA exuda-
tion, P-deprived OX.ALMT1-1 and OX.ALMT-2 lines reach
29% and 51% of the overall exudation values of WT plants
in +Al conditions (Figure 2b). Because of their abundance
and P mobilization capacity, malate, citrate, and oxalate
are the most important OAs in root exudates in plants
under P deprivation (Jones, 1998). Nevertheless, a-ketoglu-
tarate, succinate, and fumarate have also been reported to
be able to mobilize P, although to a lower extent (Kpom-
blekou-A and Tabatabai, 1994, 2003).
Exudation recovery for MALDI-MSI analysis
One of the main challenges to determine root exudation
patterns was sample preparation since the analytes must
be fixed into a solid matrix before imaging analysis with-
out disturbing their location. Gelatine-embedded tissue
has been employed to study the distribution of negatively
charged metabolites, including OAs, in A. thaliana roots
using a DMAN matrix (Ye et al., 2013), but this technique
does not allow the study of exudates. A recent work by
Sasse et al. (2019) used a MALDI plate covered with a thin
layer of ultra-pure agarose to recover the exudates from
Brachypodium distachyon roots; then they added a mix-
ture of a-cyano-4-hydroxycinnamic acid (CHCA) and 2,3-di-
hydroxybenzoic acid (DHB) as a matrix and dried the
samples before performing an untargeted MALDI-MSI
analysis that allowed them to identify exudation patterns
of unidentified metabolites. Nevertheless, the agarose pre-
sents several drawbacks and needs thorough optimization
to avoid the presence of bubbles or cracks in the agar or
sample flaking during dehydration, which can all cause
sample deformation and decreased detection of ions,
which would be detrimental to the analysis (Yang et al.,
2012). In addition, the highly complex background origi-
nated from CHCA and DHB matrices and the agarose itself
can hinder the identification of metabolites in untargeted
analysis, as was the case in the experiment mentioned. For
these reasons, we did not employ this method.
Our objective was to employ the simplest and most
effective method to collect and fix the analytes to study
exudation patterns. Briefly, plants were incubated on nylon
membranes wet with medium, attached to stainless steel
MALDI imaging plates. After 7 h of incubation and drying
at room temperature, CHCA was sprayed over the samples
before analysis in a Synaptâ (G1) HDMS in targeted HRMS/
© 2021 Society for Experimental Biology and John Wiley & Sons Ltd,
MS mode (Figure 3a). Our rationale was that secreted
molecules would slightly diffuse during the incubation, but
they would be fixed after drying the samples. Since nylon
membranes have been previously used for MALDI-MSI
through the imprinting technique (Dong et al., 2016), we
expected that the analytes would be correctly desorbed
and ionized from the matrix-covered membrane by the
laser during ionization. A similar approach has recently
been employed to fix rhizosphere compounds on PVDF
membranes and perform an indirect global MALDI-MSI
analysis taking advantage of the high resolution of 15-T
Fourier transform ion cyclotron resonance coupled with
MS to differentiate compounds (Velickovi
c et al., 2020).
Since in this study exudation was performed directly on
the membranes, this constitutes a direct analysis, targeting
OAs to achieve a high selectivity, as explained in the fol-
lowing section.
Targeted MALDI-MSI HRMS/MS method development
Targeted MALDI-MSI HRMS/MS methods were developed
to overcome the noisy signal from matrix ions and to con-
firm the identity of the analytes. Although the CHCA matrix
generates many ions below 600 m/z and can form adducts
with small molecules (Shroff et al., 2009; Silva et al., 2016;
Ye et al., 2013), we developed targeted MALDI-MSI HRMS/
MS methods aiming to study OA localization with high
sensitivity and selectivity. Because citrate and malate are
the two main root OAs exuded in response to P in most
plants (Jones, 1998; Ryan et al., 2001), they were selected
to be analyzed. Specific precursor and fragment ions were
selected from the MSI profiling analysis of spots of malate
or citrate/isocitrate on a liquid medium-saturated nylon
membrane. We generated two HRMS/MS methods
(Table S1). The [2((M- H2O)-H+Na)-H] adducts at 274.98
and 391.18 m/z for malate and citrate/isocitrate, respec-
tively, were filtered as precursors and fragmented with
optimized CE. EDC was employed to improve the detection
of 114.99 m/z for malate and 111.01 m/z for citrate/isoci-
trate; the observed ions at 115.03  0.05 and
111.05  0.04 m/z, respectively, were used to graph the
ion maps. Here, fragment 111.01, which is common to
citrate and isocitrate, was used to improve sensitivity.
However, if isomeric specificity is aimed at, their specific
fragments (87 and 73, respectively) (Erro et al., 2009) could
be employed. Furthermore, as discussed for the UPLC-
HRMS/MS quantification method, MALDI-MSI could also
be improved by employing an instrument with hyphenated
high-resolution ion mobility as an extra dimension for sep-
aration of isomers.
The MALDI-MSI HRMS/MS methods allowed the specific
detection of the analytes with higher sensitivity than the pro-
filing method. Spots of standard malate or citrate (at 0.1, 1,
10, and 50 ng) were analyzed with the profiling and targeted
The Plant Journal, (2021), doi: 10.1111/tpj.15261
 Quantification and localization of acid exudates 9

(a)
1) Plant
2) Exudate recovery 3) Sample treatment 4) Targeted MS 5) Data treatment
samples

MALDI(-) HRMS/MS

MALDI-MSI (MS/MS)
HDI (Waters)
Capillary sprayer
C
CHCA and
analyte ions
a
CHCA - -
matrix - MS/MS
- -
A. thaliana
+/- Pi Nylon membrane,7h, Laser
Normalization Vs matrix ion
MS 0.1X médium

(b) (c) Standard solution A. thaliana

Profiling MSI Optimized HRMS/MS MSI
144.052 144.052
100 100
Citrate 391.0215 Citrate 391 > 111.0493

Citrate
111.038
111.038

%
Malate 274.9655 Malate 275 > 115.0309 0 m/z 0 m/z
-0 100 200 300 400 -0 100 200 300 400
159.004
144.052 115.028
100 100

Malate
0.1 1 10 50 ng 0.1 1 10 50 ng 115.031 159.006

%
Min Max 2.0 mm %
0 m/z 0 m/z
-0 100 200 300 -0 100 200 300

Figure 3. Targeted MALDI-MSI method development.

(a) Schema of the method.
(b) Comparison between MALDI-MSI in profiling mode (left) and the optimized MALDI-MSI TOF-MRM method (right) of citrate and malate spots of standard
solution on nylon membrane.
(c) MS/MS spectra (TOF-MRM) from standard solution (left) and A. thaliana root and exudate (right) on nylon membrane.

methods to compare them (Figure 3b). The ions selected as
precursors for the HRMS/MS methods were used to obtain
the ion maps of each analyte in the profiling MSI experiment
because the [M-H] adducts were not detected. Considering
the LOD as the lowest standard concentration with detect-
able signal, the LOD of the profiling MSI method was 10 ng
for malate and 1 ng for citrate, and was rather noisy (Fig-
ure 3b, left). The sensitivity was improved by a factor of 10
with the HRMS/MS methods, as the LODs were 1 ng for
malate and 0.1 for citrate, and the noise was eliminated (Fig-
ure 3b, right). In addition, the MS spectra obtained from
these spots (Figure 3c, left) showed almost only specific
fragments of each analyte: 115.03 and 159.01 m/z for malate
and 111.04 m/z for citrate/isocitrate. Another important step
in the generation of MALDI-MS images is the normalization
of the detected signal to account for the diversity in physico-
chemical environment in the samples and inhomogeneous
matrix deposition or co-crystallization. We used the CHCA
ion 144.05 m/z ([M-CO2-H] ) both to calibrate the m/z and to
normalize the signal in MSI experiments. Another approach
to normalization is co-spraying the matrix with a labeled
internal standard. This measure is needed for quantitative
analysis with the intrinsic cost of large quantities of the
labeled molecule. Although our methods are only
qualitative, they allow the spatial localization of citrate/isoci-
trate and malate, respectively, in plant roots and in exudates,
as detailed in the following sections. Furthermore, we
obtained similar spectra with very low noise for both ana-
lytes in samples from A. thaliana (Figure 3c, right), demon-
strating the high specificity of the MALDI-MSI HRMS/MS
methods.
MALDI-MSI spatial localization of malate and citrate/
isocitrate in roots of Arabidopsis thaliana genotypes under
P deficiency
Using the MALDI-MSI HRMS/MS methods, we evaluated
the spatial localization of citrate/isocitrate and malate exu-
dation patterns in root apices of Arabidopsis seedlings
from WT (detailed in Figure 4a,b), OX.ALMT1, and
OX.STOP1 root apices. Nevertheless, quantitative compar-
isons between malate and citrate/isocitrate cannot be per-
formed since the signal intensity depends on the
desorption and ionization of each analyte. Indeed, the
lower LOD observed on spots for citrate (0.1 ng) compared
to malate (1 ng) (Figure 3b) also resulted in a higher
citrate/isocitrate signal on tissue (Figure 4a,b, Figures S6
and S7), but this does not reflect a higher abundance in
Arabidopsis roots. On the contrary, it has been observed

© 2021 Society for Experimental Biology and John Wiley & Sons Ltd,
The Plant Journal, (2021), doi: 10.1111/tpj.15261
10 David Gomez-Zepeda et al.
MALATE CITRATE
(a)
+P
-P
OX.STOP1-1
WT
OX.STOP1-1
OX.ALMT1-2
WT
OX.ALMT1-2
that their abundance in Arabidopsis roots is similar (Zieg-
ler et al., 2016).
The malate signal was observed along the root apex and
in the rhizosphere of most plants, but no apparent differ-
ence in malate distribution was found when compared to
+P and P conditions in WT nor transgenic plants (Fig-
ure S7). Nevertheless, the malate signal tends to show a
wider spread and higher intensity in the rhizosphere of
OX.ALMT1 and OX.STOP1 lines in both +P and P condi-
tions when compared to the WT (Figure 4a, Figure S7).
These observations corroborate that the overexpression of
ALMT1 and STOP1 in Arabidopsis enhances malate exuda-
tion from the roots.
After malate, citrate is considered the second-most
highly excreted OA in Arabidopsis roots. In general, no
apparent differences were found in terms of the spatial dis-
tribution of citrate exudates (Figure 4a, Figure S8). How-
ever, when the internal citrate signal is considered, the
MALDI-MSI analysis shows a clear pattern of higher citrate
accumulation in the meristematic zone and the root cap in
both WT and transgenic plants in P conditions, whereas
in +P conditions the citrate/isocitrate signal is homoge-
neously localized along the apex, including the root hairs
(Figure 4a, Figure S8).
(b) A. thaliana roots Figure 4. MALDI-MSI TOF-MRM for the analysis of
the spatial distribution of malate (275 > 115.0309 
0.05 m/z) and citrate (391 > 111.0493  0.05 m/z) in
Malate
A. thaliana roots and malate in M. polymorpha
under P deficiency.
(a) MALDI-MSI experiments showing the spatial dis-
tribution of malate and citrate in Arabidopsis roots
of WT and selected transgenic lines under P condi-
tions. Plant roots belonging to four independent
experiments, i.e., combination of a treatment (high
Citrate
or low phosphorus) and an organic acid (malate or
citrate), are shown. Top, photo of the sample before
analysis; bottom, MALDI-MSI ion map. The inten-
sity scale was set independently per experiment.
Scale bars correspond to 1 mm.
(b) Selected MALDI-MSI experiments showing the
(c) M. polymorpha region of analysis in the primary root axis and the
+P -P details of the spatial localization of malate (top,
Malate OX.ALMT1-1 in P) and citrate (bottom,
OX.ALMT1-1 in +P). Left, photo of the sample
before analysis; middle, MALDI-MSI ion map; right,
merged image. Scale bars correspond to 1 mm.
(c) MALDI-MSI single-experiment evaluation of
malate exudation of M. polymorpha gemmalings
under P conditions. Top, photo of the sample
before analysis; middle, MALDI-MSI ion map; bot-
tom, merged image. The scale bar in the upper
panel corresponds to 2 mm.
Phenotyping of the inner and outer distribution of OAs
in the root apex could play a relevant role in the design of
crop varieties better adapted to abiotic stresses. For exam-
ple, by studying Al-tolerant and Al-susceptible wheat lines
using a synchrotron-based approach, it has been shown
that both external and internal malate–Al complexation at
the root tip is linked to the tolerance mechanisms under Al
stress (Kopittke et al., 2017). The MALDI-MSI method for
malate and citrate/isocitrate imaging reported here can
support the characterization of genetic components that
generate changes at a functional level in OA distribution
and relative concentrations both within the root tissue and
in its exudates.
Application to other plant models, malate exudation in P-
deficient Marchantia polymorpha
To test the usefulness of the MALDI-MSI method in
plants other than Arabidopsis, growth of the liverwort
M. polymorpha in P conditions was evaluated for
malate exudation using a lower spatial resolution
(200 µm) than with Arabidopsis (50 µm). Marchantia
polymorpha belongs to the bryophyte lineage and is a
valuable model for studying early traits evolved in the
ancestral land plants (Bowman et al., 2017). Recently, the
© 2021 Society for Experimental Biology and John Wiley & Sons Ltd,
The Plant Journal, (2021), doi: 10.1111/tpj.15261

transcriptional and morpho-physiological responses of
M. polymorpha to P deficiency have been characterized
(Rico-Rese
ndiz et al., 2020), shedding light on the con-
servation of P stress responses in early divergent land
plants. Marchantia polymorpha encodes several ALMT1
and STOP1 homologs in its genome, and its rhizoids
respond analogously to Arabidopsis roots under P star-
vation (i.e., increase in rhizoid length and density) (Rico-
Rese
ndiz et al., 2020). Although it has been reported that
the transcription of putative malate dehydrogenase
genes decreases in P-deprived thalli of M. polymorpha
(Rico-Rese
ndiz et al., 2020), under our experimental con-
ditions, we clearly observed a stronger malate signal in
exudates of M. polymorpha gemmalings in P condi-
tions (Figure 4c). Besides P mobilization, the changes in
OA exudation could have an essential role in the estab-
lishment of beneficial interactions with soil microorgan-
isms (Bonfante and Genre, 2008). With this experiment,
we show that the MALDI-MSI method for OA analysis
can be used to study roots of other plants species than
Arabidopsis.
CONCLUSION
In conclusion, we developed two complementary methods
for the study of OAs in root exudates. The high-throughput
UPLC-HRMS/MS-based method for the identification and
quantification of six important organic acids in root exu-
dates was tested for reproducibility, recovery, and linearity,
proving excellent in all these parameters. Detection limits
were below 0.25 µM for citric, a-ketoglutaric, malic, suc-
cinic, and fumaric acid, and below 0.6 µM for lactic acid.
More importantly, this method takes only 5 min and
achieved good resolution of all peaks using a modified C18
stationary phase. Second, we present a simple procedure
to evaluate root exudation on nylon membranes to be
imaged with MALDI-MSI. Exploiting the high selectivity
and sensitivity of the HRMS/MS mode in a MALDI–quadru-
pole time-of-flight (TOF) instrument, we obtained molecu-
lar images for citric and malic acids in root tissue and
exudates. Sensitivity proved to be 0.1 ng for citric acid and
1 ng for malic acid. Selectivity was achieved despite the
natural noisy signal of the CHCA matrix using specific frag-
ment ions of the MS2 function to generate the MALDI MS
images.
The quantitative method was validated evaluating the
well-known response of root exudation in Al toxicity and,
in parallel, P deficiency. Next, overexpressing lines of
stress-responsive malate and citrate transporter genes
were assessed with respect to OA exudation quantity and
citrate/isocitrate and malate localization in P deficiency
experiments. Both methods showed an increase in malate
exudation in the overexpression lines with higher tran-
scription levels of ALMT1 compared with the WT. Further
analysis of the UPLC-HRMS/MS data showed that the
© 2021 Society for Experimental Biology and John Wiley & Sons Ltd,
The Plant Journal, (2021), doi: 10.1111/tpj.15261
Quantification and localization of acid exudates 11
increase in malate exudation upon P and Al treatments
positively correlates with the exudation of other OAs,
remarkably a-ketoglutarate, succinate, and fumarate. This
observation suggests that ALMT1 may be permeable to
other OAs besides malate. Additionally, using MALDI-MSI
HRMS/MS we were able to observe changes in the distri-
bution of citrate within the root apex of the primary root of
Arabidopsis under P deficiency, where it accumulates in
the meristematic zone and the root cap. We showed the
transferability of this method to other plants by analyzing
M. polymorpha gemmalings, showing an increase in the
exudation of malate in P conditions.
The mass spectrometry-based methods reported here
can be employed to study the release rate and spatial loca-
tion of organic acids exuded by plants to investigate physi-
ological responses, as well as to evaluate improved
varieties. For instance, these methods can be employed to
study nutritional stresses, metal toxicity, responses to
hypoxia, responses to extreme pH, and plant–microbe
interactions and for the assessment of mutants with
defects in synthesis, mobilization, and/or exudation of
OAs.
EXPERIMENTAL PROCEDURES
Reagents and substances
Unless stated otherwise, chemical reagents were obtained from
Merck–Sigma Aldrich (Toluca, Mexico). CHCA, trifluoroacetic acid
(TFA), FA, acetonitrile, and methanol (Fisher Scientific, Waltham,
MA, USA) were MS grade. Water used was ultra-pure grade from
a Milli-Q Gradient A10 instrument (Merck-Millipore, Toluca, Mex-
ico). The purity of all standards was ≥99% and stable heavy iso-
tope-labeled standards (L-malic acid-13C4 and citric acid-2,4-13C2)
presented a labeling efficiency of ≥99%, according to the provider.
Plant material and growth conditions
Arabidopsis thaliana WT (CS70000) was used in this work. ALMT1
and STOP1 genes were amplified from WT genomic DNA using
specific primers (Table S4). The transgene constructs of ALMT1
and STOP1 under control of the CaMV 35S promoter were gener-
ated using pDONORTM221 (InvitrogenTM Waltham, MA, USA) and
pFASTG02 (Shimada et al., 2010) vectors and the Gatewayâ sys-
tem (InvitrogenTM). The resulting vectors were transformed via
Agrobacterium into WT Arabidopsis plants by a modified floral
dip method (Martinez-Trujillo et al., 2004). Two independent lines
for each construct were selected for further analysis. For M. poly-
morpha, the male strain Takaragaike-1 was used.
Arabidopsis and M. polymorpha plants were grown in medium
composed of 0.19 MS salts, 10 g L 1 sucrose, 10 g L 1 agar, 500
(+P) or 10 ( P) µM KH2PO4, and 3.5 mM MES (pH = 5.7). For Al and
control treatments, medium was composed of 0.19 MS salts,
10 g L 1 sucrose, 100 µM KH2PO4, 1 mM Ca(NO3)2, 1 mM MgSO4,
1 mM CaCl2, 700 (+Al) or 0 (control) µM AlCl3, and 6 g L 1 of gellan
gum (PhytagelTM; Sigma) (pH = 4.1). Petri dishes were placed in a
growing chamber (16 h photoperiod; 23°C) in all experiments. For
qRT-PCR, UPLC-HRMS/MS, and MALDI-MSI experiments, surface-
sterilized Arabidopsis seeds or in vitro propagated M. polymorpha
gemmae were grown on +P agar plates for 8 days after sowing
12 David Gomez-Zepeda et al.
and then transferred into their corresponding fresh +P, P, +Al, or
control agar plates for 3 days as part of the preconditioning for
exudate collection. Then, the plants were transferred to the corre-
sponding liquid exudation regarding the pre-treatment for UPLC-
HRMS/MS, MALDI-MSI, or qRT-PCR experiments, as described in
the sections below.
Real time PCR
Eight days after sowing, Arabidopsis seedlings grown in +P were
transferred to P conditions for 3 days as mentioned above. Total
RNA was isolated from whole plants using TRIzol reagent (Invitro-
gen) according to the manufacturer’s instructions. RT-PCR was
performed with an Applied Biosystems 7500 real-time PCR system
using SYBR Green detection chemistry (Applied Biosystems) and
gene-specific primers. UBQ1 (At3g52590) served as internal con-
trol. The relative expression levels were computed by the 2 DDCt
method. Primer sequences are listed in the supplementary mate-
rial (Table S4).
Root system analysis
WT and transgenic Arabidopsis seeds were sown in vitro in +P or
P conditions as described above. After germination (approxi-
mately 1 day after sowing), photographs were taken every 24 h
from day 1 to 10 and the root system was analyzed using RootNav
software (Pound et al., 2013) for primary and first-order secondary
roots and ImageJ (Schneider et al., 2012) for higher-order sec-
ondary roots.
Standard solutions, calibration curves, and QCs
Standard solutions of each analyte standard were prepared in Milli
Q-grade water at 1000 ppm and stored as single-use aliquots at
20°C until use. A mixture containing all analytical standards at
500 µM was prepared on 0.19 MS medium and a calibration curve
was prepared through serial dilution at 0.1, 0.5, 1, 5, 10, 25, 50,
100, and 500 µM in 5% methanol, 95% 0.19 MS medium with 0.1%
of FA and spiked with heavy isotope-labeled standards (L-malic
acid-13C4 and citric acid-2,4-13C2) at a final concentration of 10 µM.
QCs were prepared similarly at 0.75, 1.5, 15, and 75 µM of unla-
beled standards with 10 µM heavy isotope-labeled standards.
Accuracy and precision were assessed by analyzing the calibrators
(injected three times) and QCs (injected five times). To evaluate
the matrix effect, a pool of exudates from WT A. thaliana plants
grown in +P conditions was prepared and dispatched into differ-
ent centrifuge tubes with 700 µl of exudate. These samples were
lyophilized and dissolved with resuspension solution (5% metha-
nol, 94.9% water, 0.1% FA) without or with the analytes (n = 4) at
0.4 or 4 µM and named as Pool + 0, Pool + 0.4, and Pool + 4,
respectively.
The LOD was initially estimated as the lowest calibrator with
signal-to-noise ratio above 3 and the lowest LLOQ as 3 times the
LOD. A standard solution with the analytes at the estimated LLOQ
was analyzed six times and the standard deviation (SD) was used
to calculate LOD and LLOQ as 3 9 SD and 10 9 SD, respectively.
UPLC-HRMS/MS quantification
Eight days after sowing, plants grown in +P conditions were pre-
conditioned in P, +Al, or control conditions for 3 days and used
to evaluate the exudation rates of OAs by UPLC-HRMS/MS quan-
tification. Eight groups of 10 individuals of each genotype were
placed in an individual well of a 12-well plate (Eppendorf, Mexico
City, Mexico) containing 1 ml of liquid exudation media and incu-
bated in a growth chamber for 7 h. Each exudation medium was
© 2021 Society for Experimental Biology and John Wiley & Sons Ltd,
similar in composition to its specific preconditioning medium, but
without the gelling agent, sucrose, MES, and the extra salts for
gellan gum polymerization (1 mM Ca(NO3)2, 1 mM MgSO4, and
1 mM CaCl2, for the treatments at pH = 4.1). After incubation, the
well plates were placed in an orbital shaker (100 rpm) for 7 min
and plants were removed. Exudation medium (500 ll) was recov-
ered in 1.5-ml sterile microtubes, frozen at 80°C, and lyophilized.
The dried exudates were stored at 80°C until use.
The dried exudates were dissolved by gently vortexing the sam-
ples after adding 150 µl of resuspension solvent (5% methanol,
95% water with 0.1% of FA, containing the heavy isotope-labeled
standards at 10 µM). Samples were centrifuged for 4 min at
14 000 g at 4°C and the supernatant was transferred to polypropy-
lene plates and analyzed using an Acquity UPLC (Waters Corpora-
tion) coupled to a Synaptâ (G1) HDMS with an ESI source in
negative mode. Samples from +Al treatment were analyzed also
at 1:5 dilution with resuspension solvent to allow quantification of
analytes above the upper limit of quantification (Table S3). A C18
HSS-T3 analytical column was employed (Acquity UPLC HSS-T3,
2.1 mm internal diameter, 100 mm length, 1.8 µm particle size,)
and mobile phases consisted of: A, H2O + 0.1% formic acid; and B,
methanol + 0.1% formic acid. Five microliters of sample was
injected and separated using a 4.7-min method consisting of an
isocratic step at 1% B for 0.4 min, followed by a linear gradient up
to 80% B for 2 min, a washing step at 99% B for 0.5 min, and an
equilibration step at 1% B for 1.8 min. The MS was operated in
negative ionization mode and the TOF was set in sensitivity mode
(V-ion optics mode) with a resolution of 9000 FWHM at 0.5 sec per
scan. Source conditions were as follows: capillary voltage, 2.5 kV;
cone voltage, 40; source temperature, 120°C; desolvation tempera-
ture, 400°C; cone gas flow, 0 l h 1; desolvation gas flow:
500 L h 1. The instrument was operated in targeted HRMS/MS
mode with EDC, also called TOF-MRM. In this mode, a specific ion
of each analyte (the precursor) is filtered in the quadrupole, frag-
mented in the collision cell, and orthogonally accelerated in the
pusher for the high resolution of ions in the TOF before being
detected. In EDC mode, the pusher voltage is configured to
enhance the sensitivity of one fragment ion, while still detecting
the others. In this method, the [M-H] ions of the analytes were
selected as precursors and fragmented with optimized CE, and,
whenever possible, a fragment ion was used for EDC to improve
specificity (Table S1), although EDC was not needed to detect
citrate in the range of 0.1–100 µM. Fragments with possible inter-
ferences in exudate samples (i.e., with high variability) were
excluded from quantification.
The mass spectrometer was piloted using MassLynx (v4.1).
Skyline (v20.1.0.31) (Henderson et al., 2018; MacLean et al.,
2010) was used for peak integration and quantification from cali-
bration curves using normalization against the corresponding
labeled standard for malate and citrate (Table S3, Figure S3).
Statistical analyses were performed using R. Calibrators were
analyzed in triplicate, samples in duplicate, and QCs in quintu-
plicate. The OA exudation rates in the biological replicates were
normalized by plant to allow an easier comparison with MALDI-
MSI experiments, where single plants were analyzed. Cohen’s d,
fold change values, and P-values were calculated to complement
data analysis and offer a better interpretability of the results
(Maher et al., 2013).
MALDI-MSI
Samples for MALDI-MSI analysis were placed on a nylon mem-
brane (BrightStarâ-Plus Membranes; Applied Biosystemsâ) previ-
ously saturated with +P or P liquid exudation medium and
adhered to a MALDI-MSI stainless steel plate using double-sided
The Plant Journal, (2021), doi: 10.1111/tpj.15261

tape. During method development, solutions at different concen-
trations (0, 0.1, 1, 10, and 50 ppm) of malate or citrate standards
were analyzed. One microliter of standard solution was spotted on
a membrane with +P medium. For root exudation experiments, 8
days after sowing plants grown in +P conditions were precondi-
tioned in +P or P medium for 3 days and were individually
placed over the membrane. The prepared MALDI-MSI plate was
placed in a Petri dish containing 1 ml of the corresponding liquid
exudation medium in the bottom and in contact with the nylon
membrane attached to the plaque to prevent plants from drying
(Figure 1) and incubated in a growth chamber with continuous
light for 7 h. In the cases when +P and P treatments were evalu-
ated simultaneously, two-chambered Petri dishes were used to
avoid mixture of the exudation media. Before the analysis of Ara-
bidopsis roots, the shoots were carefully removed with a scalpel.
In the case of M. polymorpha, the plants were flattened by care-
fully placing a clean MALDI-MSI metal plate over the plants and
rapidly freezing them by immersion into liquid nitrogen. Then, the
additional plate was carefully removed. Finally, the MALDI plate
with the nylon membrane and the biological sample was dried at
room temperature in a laminar flow cabinet for 20 min before
adding the MALDI matrix. CHCA matrix was prepared at
3.6 mg ml 1 in a mixture of 50% acetonitrile, 50% water + 0.1% of
TFA. A homogeneous layer of CHCA matrix was applied over the
plate using a microsprayer from an LCT-Premier mass spectrome-
ter ESI source (Waters Corporation), with a flow of 50 µl min 1
and 600 L h 1 of nitrogen as desolvation gas.
MALDI-MSI HRMS/MS analyses were performed in negative
ionization mode in a Synaptâ HDMS (G1; Waters Corporation)
with a Nd:YAG laser using an energy of 350 (arbitrary units), a fir-
ing rate of 200 Hz during 2 sec of analysis per scan and one scan
per pixel, and a spatial resolution of 50 µm for A. thaliana samples
and 200 µm for M. polymorpha. The TOF was operated in sensitiv-
ity mode (V-ion optics mode) with a resolution of 9000 FWHM.
The parameters used for optimized targeted HRMS/MS methods
are shown in Table S1. The mass spectrometer was piloted using
MassLynx (v4.1), and HDI Imaging software (v1.4; Waters Corpora-
tion) was used to create the region of interest for analyses and to
reconstruct 2D ion density maps. The CHCA ion 144.05 m/z ([M-
CO2-H] ) was used to calibrate the m/z and normalize the signal in
MSI experiments to account for any heterogeneity in the matrix
deposition. Ion maps were generated using the quadratic of the
signal, without spatial smoothing.
ACKNOWLEDGMENTS
We are very grateful to Ms. Sofia Avila-Guzma
n (Waters Corpora-
tion, Mexico) and Dr. Hernando J. Olivos (Waters Corporation,
USA) for lending us HDI software for MSI experiments. The
authors acknowledge Fe
lix Rico-Rese
ndiz for providing M. poly-
morpha gemmalings for the MALDI-MSI experiments. This work
was partly funded by Consejo Nacional de Ciencia y Tecnolog
ıa
(CONACyT), Mexico, with master’s scholarships for MF (454143)
and HRNG (455156) and a postdoctoral fellowship for DGZ
(724164).
AUTHOR CONTRIBUTIONS
LHE and JJOO designed the research; DGZ, MF, and HRNG
performed the research and analyzed the data. All authors
contributed to the writing of this paper.
CONFLICT OF INTEREST
The authors report no conflict of interest.
© 2021 Society for Experimental Biology and John Wiley & Sons Ltd,
The Plant Journal, (2021), doi: 10.1111/tpj.15261
Quantification and localization of acid exudates 13
DATA AVAILABILITY STATEMENT
All relevant data can be found within the manuscript and
its supporting materials.
SUPPORTING INFORMATION
Additional Supporting Information may be found in the online ver-
sion of this article.
Table S1. TOF-MRM parameters optimized for MALDI-MSI and
UPLC-MS quantification of organic acids.
Table S2. Average mass error in UPLC-HRMS/MS quantification
and MALDI-MSI experiments across samples of standard solutions
and A. thaliana exudates.
Table S3. Calibration curve equations, R2, and limits of detection
and quantification for the UPLC-MS/MS quantification of organic
acids.
Table S4. List of primers used in this study.
Figure S1. Tailing of the citrate peak using a HILIC-AMIDE column.
Figure S2. Chromatograms of a calibrator containing the standard
analytes at 3 µM and a root exudate sample from A. thaliana.
Figure S3. Concentrations of organic acids in pools of A. thaliana
root exudates without or with spiked standard analytes.
Figure S4. UPLC-MS/MS TOF-MRM quantification of organic acid
efflux under Al toxicity and P deficiency from A. thaliana WT and
transgenic lines using log10-transformed data.
Figure S5. Root system analysis of transgenic lines under P suffi-
ciency (+P) and deficiency ( P).
Figure S6. MALDI-MSI HRMS/MS analysis of malate in roots of
Arabidopsis transgenic lines under P sufficiency (+P) and defi-
ciency ( P).
Figure S7. MALDI-MSI HRMS/MS analysis of malate in roots of
Arabidopsis transgenic lines under P sufficiency (+P) and defi-
ciency ( P).
Figure S8. MALDI-MSI HRMS/MS analysis of citrate in roots of
Arabidopsis transgenic lines under P sufficiency (+P) and defi-
ciency ( P).
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