Plant Physiology and Biochemistry 115 (2017) 174e182

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Plant Physiology and Biochemistry

journal homepage: www.elsevier.com/locate/plaphy

Research article

Changes in ABA, IAA and JA levels during calyx, fruit and leaves
development in cape gooseberry plants (Physalis peruviana L.)


F. Alvarez-Flo
rez a, C. Lo

pez-Cristoffanini b, O. Ja

uregui c, L.M. Melgarejo a,
M. Lo
pez-Carbonell b, *
a
Department of Biology, Faculty of Science, Universidad Nacional de Colombia, Bogota
, Colombia
b
Department of Evolutive Biology, Ecology and Environmental Sciences, Plant Physiology Section, University of Barcelona, Av. Diagonal 643, 08028,
Barcelona, Spain
c
Unitat de T gics, Universitat de Barcelona, c/ Baldiri i Reixac 10-12, 08028, Barcelona, Spain
ecniques Separatives, Centre Cientcs i Tecnolo

a r t i c l e i n f o a b s t r a c t

Article history: Changes in abscisic acid (ABA), indole-3-acetic acid (IAA) and jasmonic acid (JA) content in developing
Received 14 February 2017 calyx, fruits and leaves of Physalis peruviana L. plants were analysed. Plant hormones have been widely
Received in revised form studied for their roles in the regulation of various aspects related to plant development and, in particular,
24 March 2017
into their action during development and ripening of eshly fruits. The obtained evidences suggest that
Accepted 28 March 2017
Available online 30 March 2017
the functions of these hormones are no restricted to a particular development stage, and more than one
hormone is involved in controlling various aspects of plant development. Our results will contribute to
understand the role of these hormones during growth and development of calyx, fruits and leaves in
Keywords:
Abscisic acid (ABA)
cape gooseberry plants. This work offers a good, quickly and efciently protocol to extract and quantify
Indole-3-acetic acid (IAA) simultaneously ABA, IAA and JA in different tissues of cape gooseberry plants.
Jasmonic acid (JA) 2017 Elsevier Masson SAS. All rights reserved.
Physalis peruviana
Fruit development

1. Introduction blader-like organ during fruit development and it is a protection

Physalis peruviana L., also known as cape gooseberry, is an her-
baceous plant native to the Andes that belongs to the Solanaceae
family and the second export fruit of Colombia (Fischer et al., 2007).
It is a shrub, upright, and perennial plant that grows continuously
(from 1.0 to 1.5 m in height, up to 2 m in height when plants are
staked). Both vegetative and reproductive meristems remain active
during the plant life cycle (Ramirez et al., 2013). The purplish,
spreading branches are ribbed and covered with ne hairs. Leaves
are simple, petiolated, alternate, heart shaped and highly pubes-
cent (Fischer, 2000). Flowers are bell-shaped, nodding and arise in
leaf axils. The fruit is a yellow juice berry, to about 2 cm in diameter
and 4e10 g in weight, containing inside small seeds (150e300)
(Fischer, 1995). During ripening, the fruit colour turns from green to
orange due to chlorophyll breakdown and carotenoid accumula-
tion, and their size and weight increased linearly with fruit devel-
opment (Fischer and Martnez, 1999). The calyx (or husk)
completely encloses the ripening fruit, grows to a papery and
* Corresponding author.

pez-Carbonell).
E-mail address: mlopez@ub.edu (M. Lo
against insects, birds, diseases and solar radiation (Fischer and
Ldders, 1997). Moreover, this structure represents an essential
source of carbohydrates during the rst 20 days of growth and
development (Puente et al., 2011). Cape gooseberry fruit contains
high levels of vitamins A, B-complex, C, E and K1, as well as anti-
oxidants, phytosterols, polyunsaturated fatty acids and several
essential minerals and anti-inammatory compounds (Puente
et al., 2011). This study revealed that P. peruviana is a source of
health-related compounds found in the fruit and other parts of the
plant including leaves and steams. For this reason, in Colombia this
fruit has become promissory and even with high demand in Eu-
ropean markets. This is mainly due to its unique taste, attractive
colour and shape, nutritional importance and potential medical
value. Nevertheless, still exist considerable gaps in the control of
hormonal networks among different hormones during fruit
expansion, maturation and other aspects of ripening.
Plant hormones have a key role in most physiological processes
and play a central role in the integration of diverse environmental
cues with the plant genetic program and in shaping the morpho-
logical structures. They are directly involved in fruit set, ripening
and development (Klee and Giovannoni, 2011; Seymour et al., 2013)

http://dx.doi.org/10.1016/j.plaphy.2017.03.024
0981-9428/ 2017 Elsevier Masson SAS. All rights reserved.


F. Alvarez-Flo
rez et al. / Plant Physiology and Biochemistry 115 (2017) 174e182
and sink-related processes (Roitsch and Ehness, 2000). Specially,
fruit development and maturation are physiological process
affected by external environmental conditions (including light,
moisture, and temperature) as well as internal factors (hormones).
However, interactions between developmental control genes and
hormones involved in the origin of morphological novelties are not
well understood (Khan et al., 2012). Because of the medicinal
importance of cape gooseberry, exogenous plant growth regulators
have been widely used at different developmental stages to
enhance the growth, yield and quality of its fruits (Majumder and
Mazumdar, 2001; De Jong et al., 2009; El-Tohamy et al., 2012;
Rodrigues et al., 2013; Kaur et al., 2013; Ramar et al., 2014; Albacete
et al., 2015).
Abscisic acid (ABA) modulates numerous aspects of the plant
life, including seed dormancy, embryo maturation, and plant re-
sponses to different kinds of abiotic stresses such as drought, high
temperature, chilling, and salinity (Seki et al., 2007). The ripening
process of fruits is controlled by several hormones such as ethylene,
auxins, brassinosteroids and ABA (Karppinen et al., 2013). ABA is
also an important ripening control factor since: (i) there is a sharp
increase in ABA accumulation during the onset of fruit ripening in
climacteric fruits (Buesa et al., 1994); (ii) ABA accumulates pre-
ceding ethylene release in climacteric fruits (Zhang et al., 2009);
(iii) the application of exogenous ABA enhances the production of
several metabolites involved in promoting fruit ripening (Ban et al.,
2003; Jeong et al., 2004; Giribaldi et al., 2010); (iv) in ABA-decient
tomato mutants, the fruit did not show the normal growth pattern
observed in wild type (Galpaz et al., 2008); and (v) the de-greening
stage began later in ABA decient orange mutants (Rodrigo et al.,
2003). In addition, indole-3-acetic acid (IAA) and jasmonic acid
(JA) play a crucial role in plant growth and development and IAA
have a major role in the regulation of fruit set by increasing the
levels at fruit set and growth (Dorcey et al., 2009). Earlier molecular
studies had established a role for an Auxin Response Factor (ARF) by
controlling gibberellic acid (GA) levels (Dorcey et al., 2009) and its
interaction with Aux/IAA proteins also govern the fate of fruit
initiation events (Kumar et al., 2014). JA interacts with the other
hormones in eliciting biological activity, and play a prominent role
in signaling plant defenses (Farmer and Ryan, 1990). Jasmonates
and ABA have in many cases similar functions (Kondo and Fukuda,
2001). JA is involved in many plant responses to biotic and abiotic
stresses (Wasternack, 2007), sex determination, leaf senescence
and root growth and fruit ripening (Shan et al., 2001; Concha et al.,
2013). Moreover, interactions between ABA and JA biosynthesis
have been reported (Melan et al., 1993; Brossa et al., 2011).
However, as far as we know, there is a lack of works on
endogenous changes of other plant hormones such as ABA, IAA and
JA obtained from the same extract of cape gooseberry plants. The
aim of this work was to quantify the changes of IAA, JA and ABA
concentrations in fruit, leaves and calyx of cape gooseberry during
four stages of ripening development.
2. Results and discussion
Analytical approaches to detect JA, IAA and ABA must be
extremely selective and sensitive. Liquid chromatography coupled
to mass spectrometry (LCeMS) is the most widely employed
technique for the simultaneous qualitative and quantitative
proling of multiple classes of plant hormones because of its high
sensitivity and selectivity (Du et al., 2012). LCeMS (low-resolution
mass spectrometry) may be combined with single quadrupole or
triple-quadrupole instruments (QqQ), quadrupole linear ion trap
(QqLIT). In order to better understand the organ distribution of
several hormones inuencing plant growth and development, ABA,
IAA and JA were identied by HPLC-MS/MS in MRM mode (Figs. 1
175
and 2). This technique has the main advantage of high sensitivity
and has become a versatile tool to analyze trace plant hormones.
The present work offers a good, quickly and efciently protocol to
extract and quantify simultaneously ABA, IAA and JA in different
tissues of cape gooseberry plants.
2.1. Analysis of ABA in fruits, leaves and calyx of cape gooseberry
The identication of ABA, IAA and JA both in standards and in
different tissues of cape gooseberry plants by LC-ESI-MS/MS are
presented in Figs. 1 and 2. Changes in total endogenous concen-
trations of ABA, IAA and JA in different organs of P. peruviana are
shown in Figs. 3e5. In this study, three organs (calyx, fruit and
leaves) and four stages of organ development (stage III, stage IV,
stage V and stage VI) were chosen for analysis and statistical dif-
ferences between organs and hormone concentrations were
detected.
Changes in total ABA concentrations are shown in Fig. 3. ABA
concentrations show important changes especially in fruits,
increasing ve-fold from stage III to stage IV (from 405 ng g
1, f.w.
to 2352 ng g
1, f.w.); then, once maximum values are reached, ABA
progressively decreases (to 1100 ng g
1, f.w.) in the last develop-
ment stage (stage VI). This behavior has been reported in other
works where it was found that ABA continuously changed at the
different development stages of seeds and fruits: once ABA reached
its peak, seriously physiological fruit dropping begins (Fan et al.,
2004; Liu et al., 2007). The role of ABA in inuencing fruit devel-
opment is well documented and ABA biosynthesis in plants has
been well established (Nambara and Marion-Poll, 2005; North
et al., 2007). Its biosynthesis is closely related to that of carotenoids;
therefore, we suggest that the decreasing levels of ABA are related
to the increasing orange color of ripening fruits because of a high
carotenoid biosynthesis. Nevertheless, although considerable pro-
gresses in regards to the role of ABA in the regulation of eshly fruit
ripening have been made, the molecular mechanisms still remain
to be elucidated. The ABA content of calyx is similar between stage
III and IV (around 552 ng g
1, f.w.), and remained constant between
these stages but it slightly decreases from stage IV to stage VI
(357 ng g
1, f.w.), suggesting the end of growth period and the
increase in carotenoid synthesis in this ripening tissue. It is worth
to note the purple color (results not shown) of calyx extracts before
being injected into the LC-MS/MS equipment. As already reported
(Caldwell et al., 1998), there is an increased synthesis of phenyl-
propanoids (e.g. anthocyanins) in the epidermis that absorb this
radiation and act as antioxidants. Since ABA also may have a major
role in regulation of anthocyanin biosynthesis (Jeong et al., 2004;
Loreti et al., 2008; Jia et al., 2011), the purple color observed de
visu in calyx extracts suggest us the presence of phenolic com-
pounds such as anthocyanins, which are known to have, among
others, an important antioxidant role in tissues. The endogenous
ABA content in leaves show not so remarkable changes as that
observed in fruits. On the contrary, the ABA content of young leaves
at stage III was higher (500 ng g
1, f.w.) than that observed at stage
IV (200 ng g
1, f.w.). Then, there was a small increase in ABA levels
at stage V (250 ng g
1, f.w.) and nally ABA levels drop at the end of
the development period at the same values as those of stage IV (to
188 ng g
1, f.w.). ABA is needed to modulate responses to envi-
ronmental stresses but also growth and development, particularly
seed development and fruit ripening (Zhang et al., 2009; Sun et al.,
2012). Thus, once leaves are totally expanded and the growth is
accomplished, high ABA levels are not needed, unless a stress sit-
uation appears. The dynamic balance between biosynthesis and
catabolism determines the endogenous levels of ABA; both pro-
cesses are regulated by 9-cis-epoxycarotenoid dioxygenase (NCED)
and ABA 8-hydroxylase (CYP707A), respectively. Moreover, the
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Fig. 1. Trace chromatograms for a standard solution. A) IAA; B) IAA-d5; C) JA; D) JA-d5; E) ABA; F) ABA-d6.


F. Alvarez-Flo
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Fig. 2. Trace chromatograms for a fruit sample of cape gooseberry. A) IAA; B) IAA-d5; C) JA; D) JA-d5; E) ABA; F) ABA-d6.
178

F. Alvarez-Flo
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Fig. 3. Changes of ABA concentration (ng$g
1, f.w.) in calyx, fruits and leaves of cape gooseberry plants at four stages of development (III, IV, V and VI).

Fig. 4. Changes of IAA concentration (ng$g
1, f.w.) in calyx, fruits and leaves of cape gooseberry plants at four stages of development (III, IV, V and VI).

ABA conjugation by cytosolic UDP-glucosyltransferases or release
by b-glucosidases is also important for maintaining ABA homeo-
stasis (Leng et al., 2014) and distribution throughout the plant.
2.2. Analysis of IAA in fruits, leaves and calyx of cape gooseberry
Changes in IAA levels in the three organs studied are shown in
Fig. 4 and statistical differences between them are observed. The
highest levels of IAA in leaves corresponded to young leaves at
stage III, when they were growing (500 ng g
1, f.w.). Then IAA levels
decreased at stage IV and afterwards they increased again until
leaves were adult at stage V (around 300 ng g
1, f.w.); nally, IAA
content decreased at the end of the growth period. IAA is essential
in regulating plant growth, especially when tissues are young.
Therefore, auxins (IAA) play an important role in ripening fruits
(Epstein et al., 2002). Several studies with other Solanaceae plants
like tomato showed that IAA levels changed during the fruit
maturation process (Buta and Spaulding, 1994) and so does the
capacity of the tissue to metabolize IAA (Riov and Bangerth, 1992;

et al., 1994). On the contrary, fruits and calyx showed
Catala
lower IAA levels along the four periods of development, in com-
parison to those of leaves (Fig. 4). Fruits showed progressive in-
creases of IAA from the beginning to the end of the growth period
(from 100 to 150 ng g
1, f.w.). This is in accordance with other
works suggesting that low auxin is required for the initiation of
ripening (Devoghalaere et al., 2012; Kumar et al., 2012). It has been
reported (Kumar et al., 2014) that GH3 class of proteins, which are
required for auxin conjugation, maintain physiologically active
concentrations of auxins. These proteins have been found at high
levels in mature fruits of several fruit-bearing species (Bo ttcher


F. Alvarez-Flo
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Fig. 5. Changes of JA concentration (ng$g
1, f.w.) in calyx, fruits and leaves of cape gooseberry plants at four stages of development (III, IV, V and VI).
et al., 2010). Looking at the example of their involvement in fruit
development, over-expression of a capsicum GH3 gene was found
to reduce auxin levels in tomato fruits and eventually this reduction
was thought to be responsible for the early fruit-ripening pheno-
type of these transgenic tomatoes (Liu et al., 2005). In the calyx
structure, IAA was only detected at the end of the growth period
(100 ng g
1, f.w.). Fruits presented small changes in IAA content
along the four stages of development. Nevertheless, leaves are the
organs that showed the most important changes in IAA concen-
trations. It has been reported that in eshy fruit, levels of IAA,
decline towards the onset of ripening and the application of auxins
to immature fruit can delay the ripening processes (Bo ttcher et al.,
2010). However, the mechanisms by which the decrease in
endogenous IAA concentrations and the maintenance of low auxin
levels in maturing fruit are achieved remain elusive (Bo ttcher et al.,
2010). The transcript of a GH3 gene (GH3-1), encoding for an IAA-
amido synthetase which conjugates IAA to amino acids, was
detected in grape berries Thus, GH3-1 expression increased at the
onset of ripening (termed veraison by viticulturists), suggesting that
it might be involved in the establishment and maintenance of low
IAA concentrations in ripening berries. Furthermore, this grapevine
GH3 gene responded positively to the combined application of ABA
and sucrose and to ethylene, linking it to the control of ripening
processes (Kumar et al., 2014). Levels of IAA-aspartic acid (IAA-
Asp), an in vitro product of recombinant GH3-1, rose after veraison
and remained high during the following weeks of the ripening
phase when levels of free IAA were low. A similar pattern of
changes in free IAA and IAA-Asp levels were detected in developing
tomatoes (Solanum lycopersicum Mill.), where low concentrations
of IAA and an increase in IAA-Asp concentrations coincided with
the onset of ripening in this climacteric fruit. Since IAA-Asp might
be involved in IAA degradation, the GH3 catalyzed formation of this
conjugate at, and after, the onset of ripening could represent a
common IAA inactivation mechanism in climacteric and non-
climacteric fruit which enables ripening. However, IAA concentra-
tions have also been claimed to be low and relatively constant
throughout berry development (Symons et al., 2006). Common to
all these reports is that, like in tomato, IAA levels are low at the
onset of ripening, and here dened as the last time point before the
179
start of sugar accumulation.
2.3. Analysis of JA in fruits, leaves and calyx of cape gooseberry
The total content in JA show statistical differences between the
organs studied (Fig. 5). Leaves showed a very high increase in JA
levels, rising form the stage III (4163 ng g
1, f.w.) to the stage IV
(14,702 ng g
1, f.w.) when they reach the mature stage. This fact can
suggest us that during this period of time plant needs to have a
good defensive capacity, since JA is known to have an important
role in biotic stresses when needed (Farmer and Ryan, 1992). Then,
foliar JA content progressively decreased when stages V and VI are
reached (Fig. 5). By contrast, no changes in JA concentrations were
detected in fruits along the four developmental stages and were the
lowest concentrations (Fig. 5) since JA levels remained around
180 ng g
1, f.w. Endogenous JA content in calyx tissue showed some
differences along the developmental stages. It was observed that JA
levels were high at the stage III (4019 ng g
1, f.w.) and V
(4500 ng g
1, f.w.), a little bit lower in stage IV (3000 ng g
1, f.w.)
and they nally drop at the stage VI (to 1487 ng g
1, f.w.), coinci-
dent with the end of the growth period (Fig. 3). Several works
(Gansser et al., 1997; Kondo and Fukuda, 2001; Bo ttcher et al., 2015)
have demonstrated that high levels of jasmonates are detected in
early stages of development in grapes and berries, which decrease
before the initiation of ripening and remained low during it.
However, our results showed no remarkable changes of endoge-
nous JA in cape gooseberry fruits along the different developmental
stages. In the opposite way, leaves showed an important increase in
JA between stage III and stage IV. As ABA did, JA showed an
important peak in leaves, increasing more than ve-fold the con-
tent showed in stage III. This behavior can be related to the jasm-
onate lipooxygenase activation, which is involved in ABA synthesis
from carotenoids (Melan et al., 1993). Several works have reported
the production of carotenoids, sesquiterpens and phenolic com-
pounds in ripening fruits, such as anthocyanins and tannins (Kondo
et al., 2000; Wang et al., 2008; D'Onofrio et al., 2009). This fact can
explain the purple color observed in the calyx extracts obtained
(results not shown), since most of these compounds have a
defensive role in front of, among others, UV light and biotic stress,
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F. Alvarez-Flo
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which are especially important in the rst stages of development.
Cape gooseberry is well adapted to high altitudes where there is an
increase in UV-radiation by developing several adaptation mech-
anisms. Among them, developing a dense pubescence that covers
all the green parts of the plant and an increase in leaf thickness and
specic leaf weight (Fischer and Melgarejo, 2014). Nevertheless,
natural UV-B radiation levels can have favorable effects on several
species, favoring secondary metabolism, reducing abundant vege-
tative growth and pathogen incidence (Fischer et al., 2016). The
relationship between ABA and JA biosynthesis, especially under
stress conditions, has been already reported (Bandurska et al.,
2003; Brossa et al., 2011). Moreover, there is a marked relation-
ship between JA and antioxidant compounds (Shan and Liang,
2010). Therefore, our results lead us to think that the increase in
JA could help tissues to synthesize antioxidant compounds that can
be crucial to the maintenance of redox homeostasis inside the cells.
Moreover, ABA, IAA, and JA play important roles in fruit, leaves and
calyx development and provide a better understanding of the plant
hormone crosstalk in physiological processes.
3. Experimental
3.1. Plant material
Plants of cape gooseberry (P. peruviana L., ecotype Colombia)
were grown in the experimental elds of the National University of
Colombia (Bogot
a, Colombia) on a peat substrate, under a photo-
synthetic active radiation (PAR) of 474 mmol m
2$s
1 and a tem-
perature of 20.7  C (0.5  C). Cape gooseberry growth is continuous
and both vegetative and reproductive meristems remain active
during the plant life cycle. Fruit development periods and growth
features of the selected stages in this study were the following:
stage III: green color at 45 days after anthesis; stage IV: yellowing-
green color at 50 days after anthesis; stage V: yellow color at 55
days after anthesis; and stage VI: orange color at 60 days after
anthesis.
3.2. Hormone analysis
Leaves, calyx and fruits at four developmental stages were
collected, lyophilized and stored at
80  C until hormone analysis.
Each sample was tested in triplicate. Young and old leaves from the
uppermost and lowest part of the plant respectively were taken.
Concentrations of ABA, IAA and JA were analysed by liquid chro-
matography coupled to mass spectrometry in tandem mode (HPLC-
MS/MS) according to Lo
pez-Carbonell et al. (2009) and Brossa et al.
(2011) with slight modications.
Lyophilized samples were ground in liquid nitrogen and 60 mg
of each powdered sample was extracted with 1 mL of meth-
anol:isopropanol (20:80, v/v, with 1% of glacial acetic acid) as
extraction solvent. Deuterium labelled standards of ABA and d6-
ABA were purchased from Plant Biotechnology Institute (National
Research Institute, Canada); JA, d5-JA, IAA and d5-IAA were ob-
tained from OlChemIm Ltd. (Olomouc, Czech Republic). Before
starting the extraction procedure, deuterium-labelled internal
standards (20 ng mL
1 of d6-ABA, and 50 ng mL
1 of d5-JA and d5-
IAA) were added to each of the samples and replicates. After
centrifugation (10,000 rpm for 15 min at 4  C), the supernatants
were collected and the pellets were re-extracted with 0.5 mL of
extraction solvent and centrifuged again. Then, supernatants were
combined and dried completely under a nitrogen stream and re-
dissolved in 200 mL of water/acetonitrile/acetic acid (90:10:0.05,
v/v), vortexed, centrifuged (10,000 rpm for 10 min at 4  C) and
ltered through a 0.22 mm PTFE lter (Waters, Milford, MA, USA).
Finally, 5 mL of each sample were injected into the LC-ESI-MS/MS.
Hormones were determined in three independent samples for
each treatment. Quantication was done by the creation of cali-
bration curves including each of the unlabeled analyte compounds
(ABA, JA, IAA,). Calibration curves for each analyte were generated
using Analyst software (Applied Biosystems, Inc., California, USA).
The limit of detection (LOD, S/N 3) and the limit of quantication
(LOQ, S/N 10) were also calculated with the aid of this software.
3.3. Chromatography
LCeESIeMSeMS procedure: The HPLC system consisted of an
Agilent Innity 1260 (Waldbronn, Germany) binary pump equip-
ped with a thermostated (4  C) autosampler. For the analysis of the
extracts, a Kinetex C18 column (50  2.1 mm, 2.6 mm) (Phenom-
enex, Torrance, CA, USA) equipped with a Securityguard C18 Phe-
nomenex (4  3 mm, i.d.) was used. Gradient elution was done with
water with 0.05% acetic acid (solvent A) and acetonitrile (solvent B)
at a constant ow-rate of 400 mL min-1. A linear gradient prole
with the following proportions of solvent B was applied (t, %B): (0,
2), (5, 2), (10, 100), (11, 100), (13, 2), (20, 2).
LCeESIeMS/MS system: MS and MS/MS experiments were
performed on an API 4000QTRAP mass spectrometer (ABSciex,
Concord, Ontario, Canada). All the analyses were performed using
the Turbo V source in negative ion mode with the following set-
tings: capillary voltage
4500 V, nebulizer gas (N2) 50 (arbitrary
units), curtain gas (N2) 30 (arbitrary units), collision gas High
(arbitrary units), declustering potential (DP), focusing potential
(FP), entrance potential 10 V, collision energy (CE); drying gas (N2)
50 (arbitrary units) was heated to 550  C. All the MS and MS/MS
parameters were optimized in infusion experiments: individual
standard solutions of ABA, ABA-d6, JA, d5-JA, IAA, d5-IAA, (0.1 ng mL-
1
) were infused at a constant ow-rate of 5 mL min-1 into the mass
spectrometer using a Model 11 syringe pump (Harvard Apparatus,
Holliston, MA, USA). Full scan data acquisition was performed by
scanning from m/z 100 to 800 in prole mode and using a cycle
time of 2 s with a step size of 0.1 ms and a pause between each scan
of 2 ms. In product ion scan experiments MS/MS product ions were
produced by collision-activated dissociation (CAD) of selected
precursor ions in the collision cell of the mass spectrometer and
mass analyzed using the second analyzer of the instrument. Mul-
tiple reaction monitoring (MRM) acquisition was done monitoring
each transition with a dwell time of 75 ms. Quantitation used MS/
MS (multiple reaction monitoring method, MRM). The MRM mode
was required because many compounds could present the same
nominal molecular mass, but the combination of the parent mass
and unique fragment ions was used to selectively monitor ABA, JA
and IAA in crude plant extracts. (see Table 1).
3.4. Statistical analysis
Analyses were performed taking into account normality, ho-
moscedasticity and randomness in the experimental design. Data
for the analytical determinations were subjected to a factorial
ANOVA (three-way ANOVA) where the variable to be evaluated was
Table 1
MS/MS parameters optimized and transitions used to identify and quantify in
MRMa. DP, declustering potential; CE, collision energy; CXP cell exit potentially.
MRM transitions DP (V) CXP (V) CE (V)
ABA 263.0/152.9
70
9
16
d6-ABA 269.0/159.0
75
9
16
JA 209.0/58.9
70
9
25
d5-JA 214.2/62.2
60
9
16
IAA 174.1/129.8
60
9
16
d5-IAA 178.9/134.8
50
7
17


F. Alvarez-Flo
rez et al. / Plant Physiology and Biochemistry 115 (2017) 174e182
the concentration of the hormones under the maturity stage and
the corresponding organ. The formula of the model was:
Concentration Hormones * Maturation * Organ. Mean compari-
sons were performed using Fischer HSD post-hoc test to examine if
differences were signicant at P < 0.05. Analyses were performed
with R software package (R Core Team, 2016).
Contributions
Each author has participated sufciently, intellectually or prac-
tically, in the work to take public responsibility for the content of
the article, including the conception, design, and conduction of the
experiment and for data interpretation.
FA, CLC and MLC carried out the experiments, including sample
analysis, results discussion and helped to draft the manuscript.
OJ carried out the LC-ESI-MS-MS procedure.
FA, LMM and MLC participated in the design of the study,
drafted the manuscript, and revised it critically.
Acknowledgements
The present study was nancially supported by grant BFU2012-
39527 from the Spanish Government. We thank N. Flechas (Uni-
versidad Nacional de Colombia) for support in statistical analysis.
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