Journal Pre-proof

MITOGEN-ACTIVATED PROTEIN KINASE

PHOSPHATASE 1 mediates root sensing of
serotonin through jasmonic acid signaling and
modulating reactive oxygen species

Karen Monserrat García-Valle, León Francisco

Ruíz-Herrera, Gustavo Ravelo-Ortega, Jesús
Salvador López-Bucio, Ángel Arturo Guevara-
García, José López-Bucio

PII: S0168-9452(22)00220-5
DOI: https://doi.org/10.1016/j.plantsci.2022.111396
Reference: PSL111396

To appear in: Plant Science

Received date: 29 June 2022
Revised date: 19 July 2022
Accepted date: 21 July 2022
Please cite this article as: Karen Monserrat García-Valle, León Francisco Ruíz-
Herrera, Gustavo Ravelo-Ortega, Jesús Salvador López-Bucio, Ángel Arturo
Guevara-García and José López-Bucio, MITOGEN-ACTIVATED PROTEIN
KINASE PHOSPHATASE 1 mediates root sensing of serotonin through
jasmonic acid signaling and modulating reactive oxygen species, Plant Science,
(2022) doi:https://doi.org/10.1016/j.plantsci.2022.111396
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MITOGEN-ACTIVATED PROTEIN KINASE PHOSPHATASE 1 mediates root
sensing of serotonin through jasmonic acid signaling and modulating reactive
oxygen species

Authors:

Karen Monserrat García-Vallea, León Francisco Ruíz-Herreraa, Gustavo Ravelo-

Ortegaa, Jesús Salvador López-Buciob, Ángel Arturo Guevara-Garcíac, José

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López-Bucioa *

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a
Instituto de Investigaciones Químico-Biológicas, Universidad Michoacana de San
Nicolás de Hidalgo, CP 58030 Morelia, Michoacán, México.
b
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Investigador de Cátedras CONACYT, Instituto de Investigaciones Químico-
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Biológicas, Universidad Michoacana de San Nicolás de Hidalgo. Edificio B3,
Ciudad Universitaria, Morelia, Michoacán, México.
c
Instituto de Biotecnología, Universidad Nacional Autónoma de México. Apartado
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Postal 510-3, 62250 Cuernavaca, Morelos, México.

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E-mail:
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K. M. García-Valle: mnsg.3212@gmail.com

L. F. Ruíz-Herrera: ainuropoda@hotmail.com

G. Ravelo-Ortega: gustavo.ravelo@umich.mx

J. S. López-Bucio: salvador.bucio@umich.mx

A. A. Guevara-García: aguevara@ibt.unam.mx

J. López-Bucio: jbucio@umich.mx
*Corresponding author:

José López-Bucio

e-mail: jbucio@umich.mx

Instituto de Investigaciones Químico-Biológicas, Universidad Michoacana de San

Nicolás de Hidalgo, CP 58030 Morelia, Michoacán, México.

Abstract

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Serotonin (5-hydroxytryptamine) acts as a neurotransmitter in mammals and is

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widely distributed in the plant kingdom, where it influences root growth and
defense. Mitogen-Activated Protein Kinases (MAPKs) and MAPK phosphatases

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(MKPs) play critical functions in decoding hormonal signalling, but their possible
roles in mediating serotonin responses await investigation. In this report, we
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unveiled positive roles for the MITOGEN-ACTIVATED PROTEIN KINASE
PHOSPHATASE1 (MKP1) in the inhibition of the primary root growth, cell division,
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meristem structure, and differentiation events in Arabidopsis seedlings. mkp1
mutants were less sensitive to jasmonic acid applications that halted primary root
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growth in wild-type (WT) plants, and consistently, the neurotransmitter activated

the expression of the JASMONATE ZIM-domain (JAZ) proteins JAZ1 and JAZ10,
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two critical proteins orchestrating jasmonic acid signalling. This effect correlated
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with exacerbated production of endogenous reactive oxygen species (ROS) in the

WT, a process constitutively manifested in mkp1 mutants. These data help to
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clarify the relationship between serotonin and growth/defense trade-offs, and

reveal the importance of the MAPK pathway in root development through ROS
production.
Abbreviations

COI1 – CORONATINE INSENSITIVE LOCUS1

DSP – DUAL SPECIFICITY PHOSPHATASE

GFP – GREEN FLUORESCENT PROTEIN

H2DCF-DA – 2’7’-DICHLOROFLUORESCEIN DIACETATE

JA – JASMONIC ACID

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JAZ – JASMONATE-ZIM DOMAIN

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LOX2 – LIPOXYGENASE2

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MAPK – MITOGEN-ACTIVATED PROTEIN KINASE

MAPKK – MAP KINASE KINASE e-

MAPKKK – MAP KINASE KINASE KINASE
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MKP – MAPK PHOSPHATASE

MKP1 – MAP KINASE PHOSPHATASE1

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MPK6 – MITOGEN ACTIVATED PROTEIN KINASE 6

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MS – MURASHIGE AND SKOOG

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PAMP – PATHOGEN ASSOCIATED MOLECULAR PATTERN

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PI – PROPIDIUM IODIDE

PRL – PRIMARY ROOT LENGTH

PPM – METAL-DEPENDENT PROTEIN PHOSPHATASES

PTP – PROTEIN-TYROSINE PHOSPHATASE

ROS – REACTIVE OXYGEN SPECIES

WT – WILD TYPE
Keywords: Serotonin, signal transduction, root development, Arabidopsis,
jasmonic acid.

1 Introduction
Serotonin (5-hydroxytryptamine) biosynthesis is ubiquitous in most organisms from
bacteria and archaea to eukaryots such as protists, fungi, mammals and plants.
Acting as a neurotransmitter in mammals, it modulates synaptic processes in the
central nervous system that control sleep, mood, and circadian rhythms [1-4]. In
other organisms, serotonin performs a wide variety of functions from development

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to activation of the immune system [5-9]. Particularly in plants, this molecule occurs
in most tissues, including the root system, where it antagonizes auxin signaling

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while promoting defense through the reinforcement of the cell wall [10-16]. Genes
related to jasmonic acid (JA) and ethylene signaling are necessary to modulate
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serotonin growth responses, particularly when its endogenous levels rise or upon
treatments that block cell division and elongation within the root tip. Indeed,
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changes in reactive oxygen species (ROS) appear to crosstalk with JA in
serotonin-treated roots [14], but currently, the molecular mechanisms underlying
these cellular interactions remain unclear.
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Mitogen-Activated Protein Kinase (MAPK) modules are highly conserved in

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eukaryots. These kinase cascades involve at least three elements, a MAP KINASE
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KINASE KINASE (MAPKKK), a MAP KINASE KINASE (MAPKK) and a MAP

KINASE (MAPK), activated by phosphorylation. In plants, diverse modules
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integrate a broad range of exogenous and endogenous signals, and their de-
phosphorylation implies the action of phosphatases belonging into three families:
(1) the phosphoprotein phosphatases, (2) the metal-dependent protein
phosphatases (PPM), and (3) the tyrosine phosphatases (PTP) [17-24].

The MAP KINASE PHOSPHATASE1 (MKP1) is a tyrosine phosphatase belonging

to the Dual Specificity Phosphatase (DSP) subfamily, which can dephosphorylate
the Ser/Thr and Tyr residues of the target proteins [25]. Specific regulation of target
kinases by MKP1 includes MPK3 and MPK6, which orchestrates pathogen,
hormonal, and nutrient signaling [26]. This module also operates for sensing
pathogen associated molecular patterns (PAMPs) from Pseudomonas syringae
[27, 28], and in responses to UV-B light stress [29]. The Arabidopsis mkp1 mutant
has elevated levels of the main ROS producer, the NADPH oxidase RBOHD,
which correlates with rapid increases of ROS in plants under a variety of stimuli
[30-33].

The Arabidopsis root system is useful to understand serotonin signaling owing its
simple structure and the possibility to grow the seedlings in vitro using agar plates
[14]. After germination, Arabidopsis develops a taproot system, where the primary

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root comprises the main growth axis from which lateral roots emerge and form new
branches that assist in soil exploration and water and nutrient uptake [34, 35]. The
dominant growth of the primary root occurs by the activity of the root meristem, a

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population of rapidly dividing, mitotic cells. Understanding the mechanisms behind
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root organogenesis is critical to improve plant growth, and hence agriculture in the
long term [36-38]. In this work, we investigated the participation of the MKP1
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phosphatase in the Arabidopsis root system configuration of all major zones,
including the meristem, elongation, transition and differentiation, and its relation
with the JA signaling pathway and detection of ROS using fluorescent dye 2’, 7’-
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dichlorofluorescein diacetate (H2DCF-DA), and confocal microscopy. Comparison

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of the root phenotypes in the WT and mkp1 mutants demonstrates the involvement
of this phosphatase in serotonin signal transduction, jasmonic acid-related gene
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expression, and ROS accumulation within the primary root tip.

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2 Materials and methods

2.1 Plant material and growth conditions

Arabidopsis thaliana WT (Col-0), the transgenic lines CycB1:uidA [39],
JAZ1/TIFY10A-GFP [40], JAZ10::JAZ10-GFP [41], LOX2::uidA [42], and mkp1
mutant [43, 44] were used for the experiments. Generation of
mkp1/JAZ1/TIFY10A-GFP, mkp1/JAZ10::JAZ10-GFP, and mkp1/LOX2::uidA was
done by outcrossing mkp1 with pollen from the corresponding transgenic lines to
obtain the F1 progeny and these plants were allowed to self-fertilize to get the F2
generation. Homozygous seedlings had resistance of their primary roots to
serotonin. The presence of the reporter genes in mkp1 was confirmed by confocal
microscopy and X-Gluc staining for GFP and GUS activity, respectively. The
seedlings were propagated for three generations.

All seeds were disinfected with 95% (v/v) ethanol and 20% (v/v) commercial bleach
during five minutes each, and then washed five times with sterilized deionized
water. After cold stratification (4°C) during two days, the seeds were sown on Petri
plates containing solidified 0.2X MS medium with 0.6% sucrose and 1% phytagar.

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Plates were placed in a plant growth chamber at 21°C with a photoperiod of 16h
light / 8h darkness and a light intensity of 200 µmol/m-2/s-1. After four days, the
seedlings were transferred to 0.2X MS medium containing plates, supplemented

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with different concentrations of serotonin or the corresponding treatment, and
grown for seven more days. e-
2.2 Growth analysis and measurements of cell zones
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Root growth was analyzed in plants 10 days after germination and the length of the
primary root was measured using a ruler. Then, the roots were photographed using
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a Leica DFC450C microscope with Nomarski optics. The sizes of the different cell
zones, as well as the first three cortical cells of the elongation zone were measured
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using ImageJ software (https://imagej.nih.gov/ij/). Meristematic zone was

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measured from quiescent center to the first cell in the transition zone; the
elongation zone, from the first cell starting to elongate to the first fully elongated
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one; and for the differentiation zone the distance between the end of meristematic
zone to the first root hair was taken. For each treatment, a sample of 15 seedlings
was analyzed and the experiments were replicated at least three times.

2.3 Histochemical analysis

For histochemical analysis of β-glucuronidase (GUS) activity in the transgenic lines
CycB1:uidA and LOX2::uidA, seedlings were stained and incubated overnight at
37°C in a X-Gluc reaction buffer (0.5 mg mL-1 5-bromo-4-chloro-3-indolyl-D-
glucuronide in 100 mM sodium phosphate, pH 7). The stained plants were cleared
and fixed by using the protocol described by [45], in which seedlings were
incubated for 60 min in 0.24 N HCl in 20% methanol (v/v), at 62°C. Then, the
solution was substituted with 7% NaOH (w/v) in 60% ethanol (v/v) for 20 min at
room temperature. Finally, seedlings were dehydrated with 40, 20, and 10% (v/v)
ethanol solutions, 20 min each, and then fixed with 50% glycerol (v/v). A minimum
of 9 transgenic plants per treatment were analysed using the Nomarski optics on a
Leica DM500B microscope.

2.4 ROS detection

ROS were detected using the probe 2’, 7’-dichlorofluorescein diacetate (H2DCF-

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DA). Seedlings were incubated in 10 µM dilutions of H2DCF-DA during 30 min in
darkness at room temperature, allowing the dye to penetrate into root tissues, and

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then washed three times to remove excess dye. Inside cells, H2DCF-DA is
deacetylated by cellular esterases to a non-fluorescent compound, which is later
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oxidized by ROS into highly fluorescent 2’, 7’-dichlorofluorescein (DCF). For ROS
detection, seedlings were placed on microscope slides, and the DCF fluorescence
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signals were detected with a confocal laser-scanning microscope (model BX50;
Olympus, Japan) with excitation / emission at 485 nm / 535 nm.
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2.5 Propidium iodide staining and GFP confocal imaging

Detection and quantification of GFP fluorescence was performed via confocal
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microscopy in transgenic seedlings previously stained with propidium iodide (PI; 10

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mg/ml) for 1 min, rinsed in water and mounted in 50% (v/v) glycerol on microscope
slides. Detection of GFP emission was done on an Olympus FV1200 confocal
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laser-scanning microscope; wavelengths specific for IP (568 nm excitation; 585-

610 nm emission), and GFP (488 nm excitation; 500-523 nm emission) were used
and recorded separately when necessary. The GFP final images were processed
using the ImageJ software to quantify the relative fluorescence.

2.6 Statistical analysis

All experiments were repeated at least three times with a sample of 15 seedlings
per treatment. The data were analyzed in STATISTICA software (StatSoft, 2010),
all graphs show mean values ± standard error submitted to a one-way ANOVA test
and a Tukey significance test or a student’s t test. Letters or star marks were used
to indicate means that differ significantly (P ≤ 0.05).

3 Results

3.1 Arabidopsis mutants defective on the MKP1 phosphatase show low

sensitivity to root growth repression by serotonin
Serotonin regulates the Arabidopsis immune responses, a process critically

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influenced by the mitogen-activated protein kinase phosphatase 1 (MKP1) [14, 33].
To investigate the possible role of MKP1 on serotonin signaling in the root,

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Arabidopsis wild-type (Col-0) and mkp1 four-day-old seedlings were transferred to
agar-solidified MS 0.2X media supplemented with different concentrations of
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serotonin for seven days to assess its effect on primary root growth. Comparing
with plants under control conditions, 100 µM serotonin did not significantly affect
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the primary root growth, but at 150 and 200 µM the growth of WT primary roots
decreased over 50%, and the mkp1 mutants did not manifest growth retardation.
However, we detected a reduction in the mkp1 root growth comparable to the WT
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at a higher (250 µM) serotonin concentration (Fig. 1A and B).

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Primary root growth depends on cell proliferation and elongation, both of which
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affected by serotonin [46]. Thus, we evaluated the effect of serotonin on meristem

activity through the expression of mitotic cell marker CycB1:uidA in both Col-0 and
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mkp1 genetic backgrounds during 1, 4, and 7 days after transfer to medium

supplemented with 150 µM serotonin. CycB1:uidA expression decreased in
serotonin-treated WT seedlings over time, in contrast, the mkp1 mutant keeps the
CycB1:uidA expression at all times analyzed (Fig. 2A, F). Moreover, the length of
the meristematic, transition, and elongation zones drastically diminished in the WT
through time, but not in mkp1 mutants (Fig. 2B-E). These results suggest that
MKP1 participates in serotonin signaling to repress primary root growth influencing
cell division and elongation.

3.2 MKP1 mediates serotonin-induced jasmonic acid responses

Elements of the JA signaling pathway are crucial for the plant response to
serotonin [4]. To assess if MKP1 could be an intermediary in this crosstalk, WT and
mkp1 seedlings were germinated and grown 10 days on Petri plates supplemented
with JA. We used concentrations of JA from 1-4 µM that repressed primary root
growth in WT seedlings, where the root length diminished 50% in the lowest
concentration and reached the maximum percentage of inhibition as the
concentrations increased. In these assays, mkp1 mutants showed resistance to JA
at 2 and 4 µM where the primary root keeps growing, whereas the WT primary

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roots stopped to growth (Fig. 3A and B). Next, we evaluated the expression of

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JAZ1-GFP and JAZ10-GFP depending on MKP1 in both JA and serotonin
treatments. In the WT, JAZ1-GFP expression was absent in medium without any

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regulator, and JA treatments enhanced the JAZ1-GFP expression in the nuclei of
cortical, columella and stele cells (Fig. 4A and B). Under serotonin treatment, the
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JAZ1-GFP expression pattern increased in the vascular region of primary roots and
root hairs reached the meristem (Fig. 4C). In mkp1 mutants, the JAZ1-GFP
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expression was absent in control conditions, but was induced only in the outermost
cell layer at the root tip under both JA and serotonin treatments. However, it
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remained lower than in the WT (Fig. 4D-F). The JAZ10-GFP expression in WT

plants under control conditions was not observable within the primary root tip, but it
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was noticeable in response to serotonin along the primary root (Fig. 5A-C).
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Conversely, mkp1 showed no changes of JAZ10-GFP expression in the tested

conditions (Fig. 5D-F). These results indicate that serotonin-activated JA signaling
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in the WT involves both JAZ1 and JAZ10 and their induction depends of MKP1.

3.3 Jasmonic acid but not serotonin induces the expression of the
Arabidopsis lipoxygenase 2

Chloroplast lipoxygenase 2 (LOX2) is required for wound-induced jasmonic acid

accumulation in Arabidopsis [47]. Since both serotonin and the phosphatase MKP1
act in JA signaling in the root, it was of interest to know their role in the expression
of LOX2. Hence, we evaluated the level expression of JA-inducible LOX2::uidA
transgene in leaves (Fig. 6). The JA treatments, but not serotonin, strongly induced
the activity of this promoter in the WT and mkp1 mutants. These data suggest that
serotonin and MKP1 act downstream or independently of LOX2-triggered JA
biosynthesis for regulating root growth.

3.4 MKP1 orchestrates ROS accumulation in response to serotonin

ROS are second messengers in MAPK cascade activation by JA and serotonin
[14, 48]. To evaluate if ROS production could depend of MKP1 to regulate
serotonin responses, we performed the detection and quantification of endogenous
ROS in serotonin-treated WT and mkp1 seedlings using the H2DCFDA probe. As

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previously reported [14], serotonin treatments increase ROS in primary roots of WT
seedlings, which correlated with changes in root tip structure, since the root hair

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formation zone approached the columella (Fig. 7A). Comparing with the WT, mkp1
mutants grown under control conditions had an increased ROS signal, especially in
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the outer layers of the meristem, but not in the columella and quiescent center.
There was no significant difference in mkp1 with the serotonin treatment regarding
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the control condition (Fig. 7A-C), which suggests an important function for this
phosphatase as an intermediary for ROS production induced by serotonin.
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4 Discussion
In the last few years, the research field of neurotransmitters in regulating plant
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physiological processes has been rising, for instance L-Glutamate suppresses

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auxin signaling, affects primary root growth, and promotes lateral root formation
through MAPK cascades [49-51]. GABA modulates pollen tube growth, fruit
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ripening, seed germination, and responses to biotic and abiotic stress [32],
whereas serotonin regulates root growth and branching antagonizing auxin
signalling [52, 53]. The growth of primary roots of mutants in hormonal pathways,
which display altered sensitivity to serotonin, indicated a trade-off between growth
and defence that depends of JA and ethylene [14]. Currently, how serotonin
transduces cellular signalling in plants remains unknown.

Mitogen Activated Protein Kinase modules mediate hormonal, biotic and abiotic
signals [54-57]. MKP1 in an effector of JA and ethylene pathways and regulates
ROS homeostasis [30, 58-60], through the inactivation of several kinases including
MPK3 and MPK6 in response to different signals [61-63]. Thus, we tested the
hypothesis that MKP1 may act as a critical player in serotonin signalling in the
Arabidopsis primary root. The growth of wild-type seedlings and mkp1 mutants was
determined upon transfer of seedlings to medium supplemented with serotonin and
after few days an inhibition of the primary root growth was observed in WT plants,
whereas mkp1 mutants were less sensitive to the inhibitory effects of the
neurotransmitter on primary root growth. The strong resistance of mkp1 mutants to
serotonin was manifested in the cell division, transition and elongation zones,

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which are narrowed in WT seedlings. We hypothesize that these zones may play

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important roles in serotonin sensing for indeterminate root growth that occurs in
plant through most of their life cycle.

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Our data unveiled some commonalities between jasmonic acid and serotonin
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signaling. However, the chemical identity of both compounds is very different,
serotonin is an indole-amine produced from tryptophan, whereas jasmonic acid is
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an oxylipin. The requirement of MKP1 for JA response is supported by the
phenotype of the mkp1 mutant, showing resistance to the primary root growth
inhibition caused by exogenously supplemented JA when compared to the WT. It
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was confirmed from the analysis of JAZ1-GFP and JAZ10-GFP transgenic plants.
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Consistently, mkp1 mutants were resistant to both JA and serotonin and did not
manifest the high induction of JAZ1-GFP and JAZ10-GFP expression by serotonin
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in the WT.
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The possibility of an induction of JA biosynthesis by serotonin was analysed

through evaluating the expression of the lipoxygenase LOX2 via characterization of
the reporter line LOX2::uidA, whose expression is enhanced by damage and JA
treatments. Surprisingly, uidA activity did not increase in either WT seedlings or
mkp1 mutants by serotonin indicating that the effects of this indoleamine did not
involve LOX2. The fact that an Arabidopsis mutant defective on the CORONATINE
INSENSITIVE LOCUS1 (coi1) was resistant to the growth repressing effects of
serotonin on primary root growth [14] suggests that it could act as agonist of JA
signalling rather than directly on the biosynthesis achieved by LOX2.
The role of serotonin in root organogenesis involve an increased level and
redistribution of ROS, also influenced by JA and ethylene signalling, which leads to
changes in root meristem activity [14, 46]. ROS are oxidizing molecules that in low
concentrations regulate immune responses [14, 63]. In addition, hydrogen peroxide
(H2O2) a major ROS distributed within the root tip, activated MKP1 during the
challenge by bacterial and fungal pathogen-associated molecular patterns
(PAMPs) [33]. In this work, we assessed ROS levels in both WT and mkp1
seedlings after transfer to serotonin treatment and no significant differences in

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ROS production was observed in mpk1 mutants with or without serotonin,

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indicating that MKP1 is a key regulator of serotonin-induced ROS. Perhaps, the
ROS balance within narrow cellular domains at the root tip maintains mitosis and

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cell elongation for optimal development. Our results show how the absence of
MKP1 restricts the ROS imbalance caused by serotonin, which avoids root growth
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alterations. The present work evidences MKP1 participation in regulating serotonin
signalling in the Arabidopsis root through JA-ROS crosstalk, which is important to
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understand not only the mechanisms of neurotransmitter perception in plant cells,
but also to unravel how these interesting molecules balance the growth/defense
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inputs required for plant survival and adaptation.

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Author contributions
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KMGV, LFRH, and JLB planned the experiments and analyzed data. KMGV and
JLB wrote the manuscript. GRO provided mkp1/CycB1:uidA crosses and edited the
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manuscript. LFRH provided technical support and reagents. JLB, JSLB, AAGG
designed the research and provided founding and materials. All authors read and
approved the final manuscript.

Conflicts of interest

The authors declare that they have no conflict of interest.

Acknowledgments

This work was supported by the Consejo Nacional de Ciencia y Tecnología,

México (CONACYT), grant SEP-CONACYT A1-S-34768, and the Consejo de la
Investigación Científica UMSNH grant 2.26 to JLB, and the DGAPA-PAPIIT-UNAM
grant IN209420 to AAGG.

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Figure legends

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Figure 1. Effect of serotonin in primary root growth of Arabidopsis WT and

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mkp1 mutants. 4 d-old wild-type (Col-0) and mkp1 mutant seedlings were
transferred and grown for 7 d on 0.2X MS media supplemented with serotonin. (A)
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Primary root growth in response to different concentrations of serotonin (5HT).

Bars represent means ± standard error (n=15). Different letters indicate statistical
differences following a Tukey post hoc test (P≤0.05). (B) Representative
photographs of Col-0 and mkp1 seedlings exposed to control and serotonin
treatments. Scale bar: 1 cm.
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Figure 2. Serotonin affects cell division and differentiation in the primary root
through MKP1. Wild-type (Col-0) and mkp1 seedlings expressing CycB1:uidA
were transferred and grown for 7 d on 0.2X MS medium supplemented with 150
µM serotonin. (A) Representative photographs of Col-0 and mkp1 seedlings
expressing CycB1:uidA transgene, through days 1, 4 and 7 after transfer. Scale
bar: 50 µm. (B) Meristematic zone length, (C) elongation zone length, (D) length of
differentiated cells, (E) transition zone length and (F) number of cells expressing
CycB1. Values shown represent means ± standard error (n=15). Different letters
indicate statistical differences following a Tukey post hoc test (P≤0.05).

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Figure 3. Root growth response to jasmonic acid is regulated by MKP1. Wild-

type (Col-0) and mkp1 mutant seedlings were grown on 0.2X MS media
supplemented with 1, 2 and 4 µM JA for 10 d. (A) Representative photographs of
Col-0 and mkp1 seedlings exposed to control and serotonin treatments. Scale bar:
1 cm. (B) Primary root growth in response to increasing concentrations of JA. Bars
represent means ± standard error (n=15). Different letters indicate statistical
differences following a Tukey post hoc test (P≤0.05).

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Figure 4. Comparison of JAZ1/TIFY10A-GFP expression between serotonin

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and JA treatments in Col-0 and mkp1 plants. Representative confocal images of

Col-0 and mkp1 primary roots expressing JAZ1/TIFY10A-GFP under 0 (A, D), 4
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µM JA (B, E), and 150 µM serotonin (C, F), respectively. 4 d-old seedlings were
transferred to each treatment and grown for 7 d. Scale bar: 100 µM. Images are
representative from 9 seedlings analyzed. The analyses were repeated three times
with comparable results.
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Figure 5. Serotonin induces JAZ10::JAZ10-GFP expression through MKP1.

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Representative confocal images of Col-0 and mkp1 primary roots expressing

JAZ10::JAZ10-GFP under 0 (A, D), 4 µM JA (B, E), and 150 µM serotonin (C, F),
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respectively. 4 d-old seedlings were transferred for 7 d to 0.2X MS media

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supplemented with serotonin. Scale bar: 100 µM. Images are representative from 9
seedlings analyzed. The analyses were repeated three times with comparable
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results.
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Figure 6. JA but not serotonin induces LOX2::uidA in leaves. Representative

images of Col-0 and mkp1 primary roots expressing LOX2::uidA in response to 0
(A, B), 4 µM JA (C, D), or 150 µM, 300 µM, and 450 µM serotonin (E-J). 4 d-old
seedlings were transferred for 7 d to 0.2X MS media supplemented with JA or
serotonin. Scale bar: 1 cm. Images are representative from 9 seedlings analyzed.
The analyses were repeated three times with comparable results.

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Figure 7. ROS accumulation in WT plants and mkp1 mutants treated with

serotonin. Wild-type (Col-0) and mkp1 seedlings were transferred on 0.2X MS
media supplemented with serotonin and after 7 d they were stained with 10 µM
H2DCFDA and photographed by confocal microscopy. (A) Representative images
of Col-0 and mkp1-treated roots. Scale bar: 100 µm. (B) Relative fluorescence
indicating general ROS level in the meristematic zone. (C) Relative fluorescence
indicating general ROS level in the columella. Different letters indicate statistical
differences following a Tukey post hoc test (P≤0.05). Images are representative
from 9 seedlings analyzed. The analyses were repeated three times with
comparable results.

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Author statement

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KMGV, LFRH, and JLB planned the experiments and analyzed data. KMGV and JLB wrote the
manuscript. GRO provided mkp1/CycB1:uidA crosses and edited the manuscript. LFRH provided
technical support and reagents. JLB, JSLB, AAGG designed the research and provided founding and

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materials. All authors read and approved the final manuscript.

Conflict of interest statement e-

The authors declare that they have no known competing financial interests or
personal relationships that could have appeared to influence the work reported in
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this paper.
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Highlights
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 Serotonin represses root growth in Arabidopsis seedlings affecting cell division and
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elongation.
 The MAPK phosphatase MKP1 plays a critical role in transducing the root
sensitivity to serotonin.
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 MKP1 mediates jasmonic acid root responses and gene expression under serotonin
treatments that repress root growth
 MKP1 determines ROS distribution within Arabidopsis root tips