1
Invited Expert Review
ROS signaling and stomatal movement in plant responses to drought stress and
pathogen attack
Running title: ROS signaling and stomatal movement
Junsheng Qi1, Chun-Peng Song2, Baoshan Wang3, Jianmin Zhou4, Jaakko Kangasjärvi
5, Jian-Kang Zhu 6, 7 and Zhizhong Gong1*
1State Key Laboratory of Plant Physiology and Biochemistry, College of Biological
Sciences, China Agricultural University, Beijing 100193, China
2Collaborative Innovation Center of Crop Stress Biology, Henan Province, Institute of
Plant Stress Biology, Henan University, Kaifeng 475001, China
3Key Lab of Plant Stress Research, College of Life Science, Shandong Normal
University, Ji'nan, China
4State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of
Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101,
China
5Division of Plant Biology, Viikki Plant Science Centre, Department of Biosciences,
University of Helsinki, 00014 Helsinki, Finland
6Shanghai Center for Plant Stress Biology, Shanghai Institutes for Biological
Sciences, Chinese Academy of Sciences, Shanghai 200032, China
7Department of Horticulture and Landscape Architecture, Purdue University, West
Lafayette, IN 47907, USA
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: [10.1111/jipb.12654]
This article is protected by copyright. All rights reserved. Received: February 20, 2018; Accepted: April 8, 2018
2
*Correspondence: Zhizhong Gong ([email protected])
Special Issue: Cell Signaling
Edited by: Jia Li, Lanzhou University, China
Abstract:
Stomata, the pores formed by a pair of guard cells, are the main gateways for water
transpiration and photosynthetic CO2 exchange, as well as pathogen invasion in land
plants. Guard cell movement is regulated by a combination of environmental factors
including water status, light, CO2 levels and pathogen attack, as well as endogenous
signals such as abscisic acid and apoplastic reactive oxygen species (ROS). Under
abiotic and biotic stress conditions, extracellular ROS are mainly produced by plasma
membrane-localized NADPH oxidases, whereas intracellular ROS are produced in
multiple organelles. These ROS form a sophisticated cellular signaling network, with
the accumulation of apoplastic ROS an early hallmark of stomatal movement. Here,
we review recent progress in understanding the molecular mechanisms of the ROS
signaling network, primarily during drought stress and pathogen attack. We
summarize the roles of apoplastic ROS in regulating stomatal movement, ABA and
CO2 signaling, and immunity responses. Finally, we discuss ROS accumulation and
communication between organelles and cells. This information provides a conceptual
framework for understanding how ROS signaling is integrated with various signaling
pathways during plant responses to abiotic and biotic stress stimuli.
3
INTRODUCTION
Plants face fluctuating abiotic and biotic hazards throughout their sessile lives.
Drought stress and pathogen infection are two of the most important factors that cause
extensive loss to the agricultural productivity. Increasing global warming exacerbates
the frequency of extreme weather, which further threatens the agricultural
productivity and world food security. Understanding the molecular mechanisms of
plant response and adaptation to adverse environmental stimuli is urgently required
for improving crop stress resistance and increasing yields through genetic and
molecular designing breeding. Accordingly, plants have evolved sophisticated
surveillance systems to perceive and cope with these challenges (Baxter et al. 2014;
Camejo et al. 2016).
The emergence of guard cells is a major landmark in the evolution of land plants
and their adaptation to dry environmental conditions (Umezawa et al. 2010). Stomata,
which are surrounded by a pair of guard cells, are the main gateways plants use to
efficiently take up CO2 for photosynthesis while simultaneously regulating water
transpiration. Controlling water transpiration through stomata is one of the main
strategies for plants to increase drought tolerance (Schroeder et al. 2001). The opening
and closing of stomatal aperture (i.e., stomatal movements) are regulated by turgor
pressure generated by ion and water channel proteins on the plasma membranes of
guard cells (Schroeder et al. 2001; Kim et al. 2010; Engineer et al. 2016). These
movements are tightly regulated by environmental stimuli such as water status, light
4
and CO2, as well as endogenous factors such as abscisic acid (ABA), Ca2+ and
reactive oxygen species (ROS) levels under various conditions (Schroeder et al. 2001;
Young et al. 2006). Notably, the respiratory burst oxidase homologs (RBOHs)
NADPH oxidases and the core ABA signaling components emerged at almost the
same time as guard cells in mosses during land plant evolution; these components are
not found in algae (Umezawa et al. 2010; Mittler et al. 2011; Lind et al. 2015;
Sussmilch et al. 2017). This pattern of emergence suggests that apoplastic ROS, guard
cells and ABA signaling mechanism are the co-evolutionary results of the adaptation
of land plants to dry environmental conditions (Umezawa et al. 2010; Mittler et al.
2011; Lind et al. 2015; Sussmilch et al. 2017). However, the evolution of stomata has
also opened the door for pathogen invasion (Schroeder et al. 2001; Melotto et al.
2006).
When facing different biotic and abiotic stresses, plants quickly accumulate the
common ROS as the first layer for defense (Kocsy et al. 2013; Wrzaczek et al. 2013;
Baxter et al. 2014). The term “ROS” generally refers to incompletely reduced oxygen
species, including the well-studied singlet oxygen (1O2), superoxide anions (O2∙−),
hydrogen peroxide (H2O2) and hydroxyl radicals (∙OH) (Mittler et al. 2004; Camejo et
al. 2016; Sierla et al. 2016). ROS have high chemical activity and a relatively short
half-life. Due to their inherent features, all types of ROS can damage macromolecules
such as proteins, lipids and nucleic acids, eventually leading to cell death (Petrov et al.
2015). On the other hand, ROS act as signaling molecules that modify protein
properties through the formation of covalent bonds, and they also function as major
5
inducers of programmed cell death (PCD) (Neill et al. 2002; Suzuki et al. 2012;
Baxter et al. 2014; Choudhury et al. 2017; Mittler 2017). H2O2, which has a relatively
long half-life (~1 ms in cells) and is more stable than other ROS, often acts as a
transducing signal in both cell-to-cell communication and intracellular signaling to
trigger downstream responses (Wrzaczek et al. 2013; Baxter et al. 2014; Camejo et al.
2016). In addition to ROS, reactive nitrogen species (RNS), comprising nitric oxide
(NO) and its oxidized derivatives including NO2, N2O3, peroxynitrite (ONOO−),
S-nitrosothiols and S-nitrosoglutathione (GSNO), are harmful to cells, but like ROS,
they can also serve as key regulatory signals during various cellular responses under
stress conditions (Qiao and Fan 2008; Kocsy et al. 2013; Hu et al. 2015). Both NO
and GSNO modify their target proteins through the S-nitrosylation of reactive
cysteines. The modification of proteins by either ROS or RNS can alter their activity,
stability, subcellular location or interactions with other molecules (Qiao and Fan 2008;
Kocsy et al. 2013).
As by-products of both abiotic and biotic stress, intracellular ROS are produced
in organelles such as chloroplasts, peroxisomes/glyoxysomes and mitochondria,
whereas apoplastic ROS are produced by plasma membrane-localized NADPH
oxidases, cell wall peroxidases and amine oxidases (Kadota et al. 2014; Kadota et al.
2015). These ROS activate anti-oxidative systems, which maintain proper ROS
homeostasis in the cell; however, when too many ROS are produced, ROS-associated
injury or cell death cannot be avoided (Miller et al. 2010; Suzuki et al. 2012; Baxter et
al. 2014; Choudhury et al. 2017). Major ROS-scavenging enzymes including
6
ascorbate peroxidases (APX), catalases (CAT), thylakoidal ascorbate peroxidase
(tAPX), copper-zinc superoxide dismutases (SOD) and glutathione peroxidases (GPX)
provide a highly efficient system for maintaining ROS homeostasis in various sites of
a cell under normal or stress conditions (Mittler et al. 2004). Alternative oxidases
(AOXs) are activated in response to ROS formation in mitochondrial electron
transport chain (ETC) Complex III, where they indirectly prevent excess ROS
formation (Jacoby et al. 2012). Low molecular weight antioxidants such as ascorbic
acid and glutathione, which are present in almost all organelles, are involved in ROS
quenching, primarily by providing reducing equivalents to ROS scavenging enzymes.
This process can keep ROS concentrations low, even when they are produced at a
high rate, and these enzymes can also react directly with ROS (Miller et al. 2010;
Kocsy et al. 2013; Singh et al. 2016). Plants have evolved various mechanisms to
perceive, transduce and respond to ROS signals to protect themselves from pathogen
attack and from damage caused by abiotic stresses such as drought, salinity and high
and low temperatures (Miller et al. 2010; Suzuki et al. 2012; Baxter et al. 2014;
Choudhury et al. 2017).
Plant cells have developed fine-tuned regulatory mechanisms to orchestrate
stomatal movement under drought stress and pathogen attack (Schroeder et al. 2001;
Kim et al. 2010; Arnaud and Hwang 2015), and in response CO2 levels (Young et al.
2006; Engineer et al. 2016). Both drought stress and pathogen attack can trigger the
rapid production of ROS in the apoplast, which is essential for stomatal closure (Qi et
al. 2017). Furthermore, ROS signaling can occur between cells (Mittler et al. 2011).
7
These ROS signaling pathways form a complicated network that functions in plant
responses to abiotic and biotic stress (Wrzaczek et al. 2013; Zhu 2016; Qi et al. 2017).
In this review, we will summarize recent progress in our understanding of ROS
signaling and discuss the molecular mechanisms underlying apoplastic
H2O2-mediated stomatal movement and the ROS signaling network, which function
primarily during plant responses to drought stress and pathogen attack.
APOPLASTIC H2O2 MEDIATES STOMATAL MOVEMENT DURING ABA
SIGNALING
The phytohormone ABA is synthesized in different tissues and plays various roles in
seed maturation, seed dormancy, seed germination, seedling growth and development
and the regulation of gene expression and stomatal movement in response to abiotic
stress (Finkelstein et al. 2002; Zhu 2016). Drought stress promotes the production of
ABA, which is perceived by the PYR1/PYL/RCAR family of ABA receptors (Figure
1). ABA-bound PYLs interact with clade A type 2C phosphatases (PP2Cs), which are
core negative regulators of ABA signaling, releasing their inhibition of downstream
targets and thus activating protein kinases including SnRK2.2/2.3/2.6 (OPEN
STOMATA1, OST1) and GUARD CELL HYDROGEN PEROXIDE-RESISTANT1
(GHR1) (Ma et al. 2009; Park et al. 2009; Hua et al. 2012). The activated protein
kinases phosphorylate and activate their targets via their own activity or by interacting
with downstream targets such as SLOW ANION CHANNEL-ASSOCIATED1
(SLAC1), SLAH3 (SLAC1 HOMOLOGUE3) and ALUMINUM-ACTIVATED
MALATE TRANSPORTER 12/QUICKLY ACTIVATED ANION CHANNEL1
8
(ALMT12/QUAC1). At the same time, these enzymes inhibit the inward rectifier
potassium channel KAT1, resulting in stomatal closure (Vahisalu et al. 2008; Geiger
et al. 2009; Sato et al. 2009; Geiger et al. 2010; Meyer et al. 2010; Geiger et al. 2011;
Imes et al. 2013; Brandt et al. 2015).
ROS production is induced by drought stress and ABA signaling (Sierla et al.
2016). ABA-induced H2O2 accumulation was first reported in Vicia faba and
Arabidopsis thaliana guard cells (Miao YC et al. 2000; Pei et al. 2000). The
apoplastic ROS produced by plasma membrane-localized NADPH oxidases have
been well studied (Kwak et al. 2003; Miller et al. 2009; Kadota et al. 2014; Li et al.
2014; Kadota et al. 2015). The Arabidopsis genome encodes 10 NADPH oxidases
belonging to the RBOH family (Kwak et al. 2003). NADPH oxidases have NADPH
or NADH binding sites (Suzuki et al. 2011; Kadota et al. 2015). These binding sites
transfer electrons from cytosolic NADPH or NADH to apoplastic oxygen to catalyze
the production of O2∙−, which can be converted to H2O2, either spontaneously or via
the activity of superoxide dismutases (SODs). RBOHD and RBOHF are both
responsible for ABA-mediated ROS production in guard cells (Kwak et al. 2003).
NADPH oxidase activity is tightly regulated by protein phosphorylation.
ABA-activated OST1 can directly phosphorylate serine (Ser) 13 and Ser174 at the
N-terminus of RBOHF in vitro (Sirichandra et al. 2009). OST1 interacts with BAK1
(BRI1-associated kinase 1), both of which function upstream of ROS production in
guard cells, and are inhibited by ABI1 (Shang et al. 2016). BAK1 usually forms a
heterodimer with other receptor-like protein kinases. However, The BAK1’s patterner
9
and the molecular mechanism for how BAK1 regulates ROS production and stomatal
movement are not known yet (Shang et al. 2016). Besides OST1, the calcineurin
B-like (CBL)-interacting protein kinase CIPK26 interacts with and phosphorylates the
cytoplasmic N-terminus of RBOHF, but the phosphorylation sites are currently
unknown (Drerup et al. 2013). Co-expression of CIPK26 with CBL1 or CBL9
enhances the Ca2+-dependent activity of RBOHF in HEK293T cells (Drerup et al.
2013). However, whether these phosphorylation events have any effect on RBOHF
activity remains unknown.
NADPH oxidases are regulated by other factors besides protein phosphorylation.
Numerous stresses cause phospholipase Dα1 (PLDα1) to hydrolyze membrane
phospholipids into phosphatidic acid (PA) (Zhang et al. 2009). The depletion of
PLDα1 reduces ABA-mediated ROS production in guard cells as well as stomatal
closure, and ROS production can be recovered by the addition of PA in pldα1 mutants,
suggesting that PA is crucial for ROS production (Sang et al. 2001a, 2001b; Zhang et
al. 2009). The loss of the PA binding motif in RBOHD compromises ABA-mediated
ROS production (Zhang et al. 2009). PA also binds to ABI1 (ABA INSENSITIVE 1)
and inhibits its phosphatase activity, thereby increasing ABA-promoted stomatal
closure (Zhang et al. 2004). Heterotrimeric G proteins have Gα, Gβ and Gγ subunits.
PLDα1 interacts with the Gα subunit GPA1 (Zhao and Wang 2004). GPA1 positively
regulates ABA-induced ROS production (Zhang et al. 2011). In gpa1 mutants, both
ABA-induced ROS production and Ca2+-channel activation are impaired (Zhang et al.
2011). However, exogenously applied H2O2 rescues the defects in stomatal response
10
to ABA and Ca2+-channel activation in gpa1, indicating that GPA1 functions
somewhere between ABA perception and ROS production (Zhang et al. 2011). These
findings indicate that PLDα1, its product PA and GPA1 are all required for ABA
signaling and ROS production.
Genetically speaking, during ABA-mediated stomatal regulation, ABI1 acts
upstream of ROS production, while ABI2 (another clade A PP2C) acts downstream
(Murata et al. 2001). H2O2 accumulation is not altered in the ghr1 or abi2-1 mutants,
but it is greatly reduced in the ost1 and abi1-1 mutants under ABA treatment (Murata
et al. 2001; Mustilli et al. 2002; Hua et al. 2012). Furthermore, ost1 and abi1-1
respond normally to H2O2-promoted stomatal closure, but ghr1 and abi2-1 are
insensitive to this stimulus (Murata et al. 2001; Mustilli et al. 2002; Hua et al. 2012).
ABI1 interacts with two U-box ubiquitin E3 ligases, PUB12 and PUB13, but it is
degraded by these enzymes only after interacting with ABA receptors in the presence
or absence of ABA. The pub12 pub13 double mutant accumulates more ABI1 protein
and less H2O2 in its guard cells than wild type (Kong et al. 2015), which is consistent
with the role of ABI1 in negatively regulating ROS production. ABI1 physically
interacts with, dephosphorylates and directly inhibits SLAC1 activity in Xenopus
oocytes, but its in planta function is still unresolved (Brandt et al. 2015). Unlike
pathogen-associated molecular pattern (PAMP)-triggered ROS production, in which
calcium signaling functions upstream of ROS during this process, ABA signaling
triggers ROS production. Upon ABA signaling, ROS, in turn, induce increased
cytosol Ca2+ levels, likely via GHR1, as no Ca2+ channel activity was detected in the
11
ghr1 mutant (Hua et al. 2012). Notably, flg22-induced stomatal closure is also
impaired in the ghr1 mutant, suggesting that PAMP-triggered ROS regulate stomatal
closure through the activity of GHR1 (Hua et al. 2012).
NO is another signaling molecule involved in stomatal movement (Qiao and Fan
2008; Asgher et al. 2017). In guard cells, ABA and H2O2 induce NO production,
which requires nitrate reductase (NR). Indeed, the NR double mutant nia1 nia2 fails
to produce NO in guard cells under exogenous ABA or H2O2 treatment (Bright et al.
2006). ABA-induced NO production is also greatly reduced in the atrbohD/F double
mutant (Bright et al. 2006) and in the guard cells of pldα1 (Zhang et al. 2009). These
findings indicate that ABA-induced apoplastic H2O2 production is required for NO
biosynthesis. ABA-induced NO can modify the reactive cysteine thiol in a protein to
form S-nitrosothiol. Protein S-nitrosylation is important for protein activity and
stability (Hu et al. 2015). OST1 activity is abolished by S-nitrosylation at cysteine
(Cys) 137, which is adjacent to the kinase catalytic site (Wang et al. 2015a),
suggesting that NO regulates ABA signaling via a feedback circuit.
As mentioned above, the production of extracellular ROS by NADPH oxidases
represents an evolutionary achievement for land plants (Mittler et al. 2011). More
importantly, apoplastic ROS may act as signaling molecules to transduce extracellular
signals into cells, which protect the inner components of the cells from ROS damage.
The plasma membrane-localized LRR receptor-like protein kinase GHR1 is a key
component in apoplastic ROS signal transduction (Hua et al. 2012). GHR1 directly
interacts with and activates the slow-type anion channel SLAC1 (Hua et al. 2012). It
12
is likely that OST1 phosphorylates and activates RBOHF to produce apoplastic ROS,
a process inhibited by ABI1 (Yoshida et al. 2006). Meanwhile GHR1 transduces
apoplastic ROS signals into guard cells via an unknown mechanism that is
specifically inhibited by ABI2 (Hua et al. 2012). The N-terminal extracellular domain
of GHR1 contains three Cys residues, including a Cys pair (Cys-57, Cys-66) that is
well conserved in most RLKs and is required for their normal functioning (Hua et al.
2012). However, the other Cys residue, Cys-381, does not function in ROS sensing, as
its mutation to Ala does not affect the role of GHR1 (Hua et al. 2012). Thus, whether
GHR1 or other plasma membrane receptor-like protein kinases can perceive
apoplastic ROS and transduce them inside the cell remains unknown. Perhaps GHR1
interacts with oxidized, ROS induced peptide ligands or another RLK that can be
induced or oxidized by ROS to transduce apoplastic ROS signals. Recent studies
suggest that members of the large family of cysteine-rich receptor like kinases (CRKs)
are important candidates that may achieve extracellular ROS signal transduction to
downstream targets (Idanheimo et al. 2014; Bourdais et al. 2015). Some CRKs play
general and/or specific roles in pathogen- and biotic stress-induced stomatal closure
(Idanheimo et al. 2014; Bourdais et al. 2015). Given the high similarity among these
CRKs and the large family size, additional studies examining their roles in H2O2
signal transduction are required using the newly developed CRISPR/Cas9 technique
to create multiple mutants (Wang et al. 2015b).
As we discussed above, apoplastic ROS might be sensed by some plasma
membrane proteins to transduce signals into cells. Given that RBOHD is a membrane
13
protein, it undergoes endocytosis, which is cooperatively regulated by clathrin- and
microdomain-dependent endocytic pathways (Hao et al. 2014). ABA and flg22
treatments increase monomer-dimer transitions and the diffusion efficiency of
RBOHD (Hao et al. 2014). This process allows ROS to be generated within
endosomes for signal transduction (Hao et al. 2014). On the other hand, apoplastic
H2O2 can be transported into the cytosol by aquaporin proteins to mediate stomatal
movement. ABA-activated OST1 phosphorylates Ser121 of the aquaporin protein
PIP2;1, which mediates water transport and also likely moves H2O2 from the apoplast
to the cytosol (Grondin et al. 2015; Rodrigues et al. 2017). The pip2;1 mutant is
defective in both ABA-induced ROS accumulation and ABA-promoted stomatal
closure (Grondin et al. 2015). As an oxidative chemical, cytosolic H2O2 can directly
oxidize some proteins to affect their activity. For examples, H2O2 oxidizes the general
ROS-scavenging protein ATGPX3, which physically interacts with ABI2, thereby
converting it from the reduced form to the oxidized form, which significantly
decreases ABI2 activity (Miao et al. 2006). As a ROS scavenger, in turn, ATGPX3
can scavenge ABA- or drought-induced H2O2. Thus, ATGPX3 may act as a ROS
sensor to transduce oxidative signals during ABA and drought stress signaling (Miao
et al. 2006). Furthermore, cytosolic H2O2 may in turn inhibit ABA signaling as both
ABI1 and ABI2 are sensitive to H2O2 in vitro (Meinhard and Grill 2001; Meinhard et
al. 2002). It is also found that CPK8 interacts with cytosolic CAT3 and positively
regulates its activity by phosphorylating Ser261 during ABA-mediated stomatal
regulation (Zou et al. 2015). Both the cpk8 and cat3 mutants have lower catalase
14
activity, accumulate more H2O2 and lose water more rapidly than wild type (Zou et al.
2015). These findings suggest that apoplastic ROS communicate with the cytosol to
regulate ABA signaling and stomatal movement.
CROSSTALK AMONG CO2, ABA AND ROS SIGNALING DURING
STOMATAL MOVEMENT
Elevated atmospheric CO2 levels on Earth are causing global warming, which
increases the severity of drought stress (Xu et al. 2015). However, high concentrations
of CO2 provide more carbon for photosynthesis and stimulate plant growth under
favorable water and nutrient conditions (Xu et al. 2015). Plants have evolved ways to
optimize stomatal activity to efficiently acquire CO2 for photosynthesis while
reducing water loss via transpiration (Chater et al. 2013). Thus, guard cells have
developed a fine regulatory system for responding to atmospheric CO2 levels and
integrating information about environmental CO2 levels with endogenous ABA
signaling (Kim et al. 2010). Short-term exposure to elevated CO2 levels provokes
stomatal closure, and long-term exposure reduces stomatal density, both of which
improve water use efficiency and protect plants against drought stress. However,
these changes can lead to increased leaf temperature, which can damage cells
(Medlyn et al. 2001). Nevertheless, treatment with high concentrations of bicarbonate
(the product of CO2 catalyzed by carbonic anhydrase, CA) induces H2O2 production
and promotes plasma membrane-localized NADPH oxidase-dependent stomatal
closure, while low CO2 levels promote stomatal opening (Kolla et al. 2007),
suggesting there is crosstalk between CO2 signaling and ROS.
15
In order to identify the components in CO2 signaling, a genetic screening using
thermal imaging to detect the temperature change based on water transpiration has
been established (Hashimoto et al. 2006). The first identified mutant by this thermal
imaging screening is the high leaf temperature1 (ht1), which showed higher leaf
temperatures and smaller stomatal apertures compared to wild type (Hashimoto et al.
2006). HT1 encodes a putative MAPKKK kinase that negatively regulates stomatal
responses to changes in CO2 levels but does not affect ABA or blue light signaling
(Hashimoto et al. 2006). A recent study indicates that βCA1 and βCA4 function in the
early CO2 response; mutations in these genes strongly impair the stomatal CO2
response and increase stomatal density, but like HT1, they do not affect responses to
ABA or blue light (Hu et al. 2010). The ca1 ca4 ht1-2 triple mutants exhibit a similar
CO2 response as ht1-2, suggesting that HT1 acts downstream of βCA1 and βCA4 in
CO2 mediated stomatal movement. βCA1 is localized to the chloroplast (Fabre et al.
2007; Hu et al. 2010). βCA4 is targeted to the plasma membrane and interacts with
PIP2;1, which might transport apoplastic CO2 and efficiently channel it to βCA4
(Wang et al. 2016).
The anion channel SLAC1 plays a crucial role in stomatal movement during CO2
signaling (Negi et al. 2008). SLAC1 can be activated by elevated intracellular
bicarbonate levels in guard cells (Xue et al. 2011). A recent study found that the
SLAC1 transmembrane domain, but not the N-terminus or C-terminus, responds to
CO2, but not to ABA, while the N-terminus is important for ABA signaling
(Yamamoto et al. 2016). Both OST1 and GHR1 can activate SLAC1 in oocytes
16
(Geiger et al. 2009; Hua et al. 2012), which can be inhibited by HT1 (Horak et al.
2016). Interestingly, the inhibition of OST1-, GHR1-activated SLAC1 by HT1 can be
counteracted by MPK12 (Horak et al. 2016). In a recent natural variation study,
MPK12 was also identified as a key player in Arabidopsis Cvi-0 Accession for guard
cell CO2 signaling (Jakobson et al. 2016). These findings suggest that MPK12 inhibits
HT1, and HT1 inhibit the OST1/GHR1-activated SLAC1. Different from HT1 that
mainly functions in CO2 signaling, MPK12 is also activated by ABA or H2O2
(Jammes et al. 2009). Interestingly, co-expressing βCA4 and PIP2;1 with
OST1-SLAC1 or CPK6/23-SLAC1 in oocytes activates SLAC1 via extracellular CO2
(Wang et al. 2016). These findings suggest that in the presence of various protein
kinases, SLAC1 directly senses cytosolic CO2/HCO3− that has been transported by
PIP2;1 and converted by βCA4 in oocytes.
The crosstalk between ABA and CO2 signaling is further supported by the
finding that ABA response mutants such as abi1-1, abi2-1, gca2, ost1 and ghr1 and
the ABA receptor mutant pyr/acars exhibit reduced guard cell responses to elevated
CO2 (Webb and Hetherington 1997; Young et al. 2006; Xue et al. 2011; Merilo et al.
2013; Horak et al. 2016). ABA biosynthesis is also required for CO2 signaling (Chater
et al. 2015). These findings suggest that full CO2 responses require components of the
ABA signalosome. It is likely that the ABA and CO2 signaling pathways converge to
mediate stomatal movement, while OST1 and GHR1 act downstream of this
convergence site (Engineer et al. 2016; Horak et al. 2016) (Figure 2). CO2-induced
stomatal closure and reduced guard cell density require ROS production by the
17
NADPH oxidases RBOHF and RBOHD. Both CO2-induced stomatal closure and
reduced guard cell density are impaired in the rbohD rbohF double mutant, ABA
deficiency mutants and high-order ABA receptor pyl mutants (Chater et al. 2015).
Silencing of OST1 and RBOH1 in tomato reduces H2O2 and NO accumulation in
response to elevated CO2 levels, while silencing NR only reduces the accumulation of
NO. These findings indicate that OST1 and NADPH oxidase-dependent H2O2
production act upstream of NO production during CO2 signaling in guard cells (Shi et
al. 2015). Together, these findings suggest that ABA and ROS signaling are required
to enhance high CO2-induced stomatal closure.
APOPLASTIC H2O2 MEDIATES STOMATAL MOVEMENT IN PLANT
RESISTANCE TO DISEASES
As mentioned above, land plants have evolved stomata for H2O transpiration and gas
exchange, which has simultaneously opened the door for pathogen invasion (Melotto
et al. 2006). Plants have evolved functional immune systems for defenses against
pathogens. The first layer of plant innate immunity is the recognition of PAMPs by
pattern recognition receptors (PRRs), which initiates PAMP-triggered immunity (PTI)
to activate downstream immune responses (the second layer), leading to a transient
increase in cytosolic Ca2+ levels, a ROS burst, stomatal closure and increased
expression of pathogen-related genes, thus restricting pathogen entry (Lu et al. 2010;
Zhang et al. 2010). ROS bursts produced by NADPH oxidases or apoplastic enzymes
can lead to rapid PCD in a few distinct cells at the infection sites, a process known as
the hypersensitive response (HR). This process can impede the invasion of biotrophic
18
pathogens because they obtain nutrients from living cells, while the neighboring cells
acquire the ability to prevent cell death through the spreading of ROS (Wu et al.
2014). Apoplastic ROS play crucial roles in mediating callose deposition and cell wall
crossing-linking, which reinforce the cell wall to impede the penetration of pathogens
(O'Brien et al. 2012).
Numerous PRRs localized on the plasma membrane include receptor-like kinases
(RLKs) and receptor-like proteins (RLPs) (Dou and Zhou 2012). In the model plant
Arabidopsis, the best-characterized RLKs include FLAGELLIN SENSING2 (FLS2)
and elongation factor-Tu receptor (EFR), which recognize the bacterial flagellin
epitope flg22 and bacterial elongation factor-Tu (EF-Tu) epitope elf18, respectively.
The perception of flg22 by FLS2 or elf18 by EFR induces their rapid association with
the co-receptor BAK1. FLS2/EFR and BAK1 represent the first layer of pathogen
signal perception required to trigger an oxidative burst within the plant (Figure 3). By
promoting stomatal closure, FLS2 plays an important role in restricting bacterial entry
into the plant (Melotto et al. 2006).
In the FLS2 signaling pathway, BOTRYTIS-INDUCED KINASE1 (BIK1) play
important role in mediating ROS production from ROBHD (Kadota et al. 2014; Li et
al. 2014). FLS2/EFR and BAK1 constitutively associate with their downstream target,
BOTRYTIS-INDUCED KINASE1 (BIK1), which is rapidly phosphorylated by
BAK1 and released from the FLS2/EFR-BAK1 complex after flg22/EF-Tu is
perceived by FLS2/EFR (Lu et al. 2010; Zhang et al. 2010). BIK1 belongs to the
RLCK-VII subfamily, which consists of 46 members (Zhang et al. 2010). Both FLS2
19
and BIK1 interact with RBOHD (Li et al. 2014). After FLS2 perceives flg22, the
activated BIK1 directly phosphorylates RbohD at Ser39 and Ser343 in a
calcium-independent manner and regulates RBOHD (Kadota et al. 2014; Li et al.
2014). Consistently, bik1 mutants are compromised in their ability to generate a rapid
ROS burst and immune responses (Lu et al. 2010; Zhang et al. 2010). However,
BIK1-mediated phosphorylation is required but not sufficient for RBOHD activation
(Li et al. 2014). The BIK1 homologs PBL1, PBL2 and PBL5 are also involved in the
generation of a pathogen-triggered ROS burst (Zhang et al. 2010; Liu et al. 2013).
CPK28 was found to interact with and phosphorylates BIK1 and contributes to its
turnover, resulting in decreased RBOHD-dependent ROS production (Monaghan et al.
2014). The mutation of CPK28 protein increases BIK1 stability and enhances
PAMP-triggered signaling and antibacterial immunity in Arabidopsis (Monaghan et al.
2014). Furthermore, a recent study indicated that the non-canonical heterotrimeric Gα
protein XLG2 interacts with both FLS2 and BIK1 and functions together with the Gβ
protein AGB1 and the Gγ proteins AGG1/2 to attenuate proteasome-mediated
turnover of BIK1 prior to flg22 perception (Liang et al. 2016). After the perception of
flg22 by FLS2, XLG2 dissociates from AGB1; its N-terminus is phosphorylated by
BIK1, which enhances flg22-induced ROS production, likely through RBOHD (Liang
et al. 2016). Similar with ABA signaling, the plasma membrane intrinsic proteins
AtPIP1;4 and AtPIP2;1 transport apoplastic H2O2 to the cytoplasm, thus activating
systemic acquired resistance and PTI (Tian et al. 2016; Rodrigues et al. 2017).
Besides BIK1 and its homologues, the calcium-dependent protein kinases CPK4,
20
CPK5, CPK6 and CPK11 are thought to be involved in the generation of a
PAMP-induced ROS bursts in Arabidopsis (Boudsocq et al. 2010; Dubiella et al.
2013). It is likely that CPK5 specifically phosphorylates RBOHD at Ser148 and
regulates its activity (Dubiella et al. 2013; Li et al. 2014). BR-SIGNALING
KINASE1 (BSK1) is a positive regulator of brassinosteroid signaling and a substrate
of the brassinosteroid receptor kinase BRASSINOSTEROID INSENSITIVE1 (BRI1)
(Tang et al. 2008). Some other factors such as BSK1 are found to interact with FLS2
and required for flg22-induced ROS bursts (Shi et al. 2013).
Besides apoplastic ROS, pathogen infection also induces the production of NO,
which is required for plant resistance to pathogen invasion (Delledonne et al. 2001).
NO production likely acts downstream of H2O2 in the plant pathogen resistance
response (Arnaud and Hwang 2015). NO modifies the Cys890 of RbohD through
S-nitrosylation, which impairs its ability to bind the cofactor FAD, thus blunting
NADPH oxidase activity (Yun et al. 2011). By contrast, S-nitrosylation at the Cys32
of cytosolic APX1 enhances its enzymatic activity for scavenging H2O2 (Yang et al.
2015). Thus, H2O2 and NO form a feedback regulation circuit in plant disease
resistance.
In addition to producing extracellular ROS via NADPH oxidases, plants can
produce these compounds via apoplastic peroxidases (Torres et al. 2002; Bindschedler
et al. 2006). In Arabidopsis, knockdown of the class III apoplastic peroxidase genes
PRX33 and PRX34 reduces ROS production and callose deposition during various
PTI responses (Daudi et al. 2012). The cytokinin RESPONSE REGULATOR2
21
(ARR2) directly regulates PRX33 and PRX34 expression, thereby mediating ROS
accumulation, stomatal closure and PTI, which are independent of ABA signaling
(Arnaud et al. 2017). As NADPH oxidases are activated by ROS, NADPH oxidases
and apoplastic peroxidases likely affect each other in the modulation of ROS bursts
during the plant immunity response.
Not surprisingly, pathogens have developed an efficient evasion system to disturb
host immunity through secreting various effector proteins, which hijack plant proteins,
as well as toxins (such as coronatine produced by Pseudomonas syringae), causing
stomata to reopen (Melotto et al. 2006; Qi et al. 2017). Under dry environmental
conditions, most phyllosphere microbes maintain low populations, and only few
bacterial pathogens enter through stomatal pores. Under high-humidity conditions, the
phyllosphere pathogens aggressively propagate and invade plants through the stomata,
causing disease breakout (Xin et al. 2016). ABA can enhance disease resistance by
closing stomatal pores (Melotto et al. 2008), but it can also increase a plant’s
susceptibility to some powdery mildew, fungal and bacterial diseases (Bostock et al.
2014). For example, abi1-1 dominant mutants and plants overexpressing
HYPERSENSITIVE TO ABA1 exhibit increased callose deposition and resistance to
bacteria P. syringae (de Torres-Zabala et al. 2007). The ABA-deficient tomato mutant
sitiens accumulates more H2O2 at the site of infection and is more resistant to the
necrotrophic fungus Botrytis cinerea than wild type (Asselbergh et al. 2007). By
contrast, the ABA biosynthetic mutants aba1-3 and ABA insensitive mutant abi1-1
exhibit reduced callose deposition and disease resistance in response to another
22
necrotrophic fungus, Leptosphaeria maculans (Kaliff et al. 2007). Some common
signaling proteins, such as OST1, GHR1 and SLAC1, are involved in both ABA- and
pathogen-mediated stomatal movement (Melotto et al. 2008; Hua et al. 2012; Deger et
al. 2015). These findings suggest that ABA plays multifaceted roles in resistance to
various diseases (Ton et al. 2009).
ROS SIGNALING IN CHLOROPLASTS, MITOCHONDRIA AND
PEROXISOMES UNDER DROUGHT STRESS AND PATHOGEN ATTACK
Under both abiotic and biotic stress, ROS are produced in various organelles due to a
metabolic imbalance. Like apoplastic ROS, these ROS can serve as stress signals to
modify some proteins and activate the expression of stress-associated genes, which in
turn counteracts stress-associated oxidative stress (Mittler et al. 2004; Miller et al.
2010; Shapiguzov et al. 2012; Suzuki et al. 2012; Singh et al. 2016). The chloroplast
is a key organelle for sensing environmental signals such as high light, low or high
temperature, salt and drought stress, as well as pathogen invasion. This organelle
produces most of the ROS found in leaf cells (Figure 4). Increased ROS production in
chloroplasts negatively affects photosynthetic electron transport, impairs the assembly
and repair of photosystem II (PSII) and affects chloroplast development (Dietz et al.
2016). Chloroplasts mainly produce singlet oxygen (1O2) in PSII and its
light-harvesting antennae (Shapiguzov et al. 2012). Although 1O2 is a ROS, it is
unusual in that it is produced under high-intensity light when the excitation energy of
triplet chlorophyll molecules is transferred to triplet-state molecular oxygen
(Triantaphylides and Havaux 2009). The Arabidopsis fluorescent (flu) and chlorina1
23
(ch1) mutants specifically accumulate 1O2 without significant coproduction of other
ROS under certain conditions (Meskauskiene et al. 2001; Ramel et al. 2013), making
them particularly useful for studying 1O2 activity. As demonstrated using these
mutants, 1O2 is crucial for regulating the expression of nucleus-encoded
stress-responsive genes, and it also promotes cell death (Wagner et al. 2004; Lee et al.
2007; Ramel et al. 2013). Carotenoids are the main 1O2 quenchers in the chloroplast.
The oxidized carotenoid product, β-cyclocitral, specifically induces the expression of
genes in the 1O2 signaling pathway but has little effect on the expression of
H2O2-mediated genes (Ramel et al. 2012). The executer1 (ex1) mutation suppresses
the 1O2-induced cell death phenotype of the flu mutant (Wagner et al. 2004) but does
not affect the accumulation of 1O2. Furthermore, crossing flu with the ex1 ex2 double
mutant fully suppressed the upregulation of almost all 1O2-responsive nuclear genes in
this mutant (Lee et al. 2007). These findings suggest that chloroplast-localized EX1
and EX2 are crucial transducers or sensors for 1O2-induced gene expression and cell
death.
The application of flg22 to Arabidopsis quickly induces specific Ca2+ transients
in the chloroplast stroma; this process relies on the presence of the
thylakoid-associated calcium-sensing receptor (CAS) (Nomura et al. 2012). CAS
regulates the PAMP-induced expression of defense genes and inhibits
chloroplast-mediated transcriptional reprogramming, likely through an 1O2-mediated
pathway (Nomura et al. 2012). These findings suggest that Ca2+ signaling
communicates with ROS signaling in the chloroplast to mediate nuclear gene
24
expression.
Pathogens can hijack plant immune signaling by secreting various effector
proteins. Pseudomonas syringae DC3000 effectors rapidly inhibit photosynthesis by
reprogramming the expression of nucleus-encoded chloroplast-targeted genes, thus
preventing the chloroplastic ROS burst. This phenomenon coincides with
pathogen-induced ABA accumulation; exogenous application of ABA suppresses
plant immunity and PSII activity (Zabala et al. 2015). The cysteine protease effector
HopN1 directly targets PsbQ in PSII, thereby mediating the degradation of PsbQ and
interfering with PSII activity, thus promoting 1O2 production (Rodriguez-Herva et al.
2012).
PSI mainly produces O2∙−/H2O2 when photosynthetic electron transfer and CO2
fixation rates are altered, especially under water-stress conditions that reduce CO2
uptake due to stomatal closure (Shapiguzov et al. 2012). Overexpressing tAPX
reduces H2O2 levels but increases 1O2-mediated stress responses in the flu mutant,
suggesting that H2O2 has an antagonistic effect on 1O2 signaling in chloroplasts (Laloi
et al. 2007). H2O2 accumulates in the chloroplasts of bundle sheath cells under high
light and induces the ABA biosynthesis-dependent expression of ROS-responsive
genes (Galvez-Valdivieso et al. 2009). H2O2 activates the kinase OXIDATIVE
SIGNAL-INDUCIBLE1 (OXI1), which is required to fully activate MPK3 and MPK6
for ROS production under various conditions (Rentel et al. 2004; Petersen et al. 2009;
Liu et al. 2010). The oxi1 mutant is more tolerant to photo-induced oxidative damage
and cell death than wild type (Shumbe et al. 2016). OXI1 also suppresses
25
ch1-mediated 1O2 production and PCD under high-light conditions independently of
EX1 and EX2, but likely through jasmonate signaling. These findings suggest that
FLU- and CHL-mediated 1O2 production regulate cell death via different pathways
(Shumbe et al. 2016).
The metabolites produced in chloroplasts under high light and various stress
conditions play important roles in ROS signaling and the regulation of nuclear gene
expression (Zhu 2016). Phosphonucleotide 3′-phosphoadenosine 5′-phosphate (PAP)
accumulates in cells under drought and high-light stress and is regulated by the
phosphatase SAL1, which dephosphorylates PAP to adenosine monophosphate (AMP)
(Estavillo et al. 2011). SAL1 activity is inhibited by oxidative stress through
oxidation-mediated dimerization, the formation of intramolecular disulfide bridges
and glutathionylation, resulting in the accumulation of PAP (Chan et al. 2016). PAP
from chloroplasts to cytosol is thought to inhibit nuclear 5′-to-3′ exoribonucleases
(XRNs), which might alter the levels of stress-responsive mRNAs via RNA cleavage
or affect transcription termination (Estavillo et al. 2011). Thus, SAL1 might be a
conserved, general ROS sensor in chloroplasts that functions in plants under both
drought stress and high-light conditions (Chan et al. 2016).
Notably, the sal1 mutation, exogenous manipulation of PAP and the xrn2 xrn3
double mutations all restore ABA responses in the ABA-hyposensitive mutants ost1,
the snrk2.2/2.3/2.6 (ost1) triple mutant and abi1-1 during stomatal movement and/or
seed germination (Pornsiriwong et al. 2017). ABA-activated SnRK2.2/2.3/2.6 kinases
phosphorylate XRN2 and XRN3 (Wang et al. 2013b), suggesting that
26
SAL1-PAP-XRN retrograde signaling can bypass the ABA pathway (Pornsiriwong et
al. 2017), but is regulated by ABA signaling (Wang et al. 2013b). SAL1-PAP-XRN
signaling positively regulates the expression of many genes involved in the ABA and
Ca2+ signaling pathways. These observations emphasize the tight communication and
complementary interactions between ROS-mediated SAL1-PAP-XRN signaling and
ABA signaling under drought and high-light conditions (Pornsiriwong et al. 2017).
Many stress treatments can quickly activate MAP kinase cascades, including
MAP3 and 6 (Meng and Zhang 2013). MPK3/6 interact with and phosphorylate ERF6
at Ser266 and Ser269, making ERF6 more stable, allowing it to directly upregulate
the expression of ROS-responsive and defensin genes and conferring fungal disease
resistance (Wang et al. 2010; Meng et al. 2013). These responses are abolished in the
tpt1 and tpt2 mutants, which lack chloroplast membrane-localized triose phosphate
translocators (TPT) (Vogel et al. 2014). Consistent with this role, the erf6 mutant
accumulates more H2O2 and is more sensitive to photoinhibition than wild type
(Wang et al. 2013a). These observations suggest that ROS are involved in regulating
the signaling of metabolites in chloroplasts that serve as retrograde signals to
coordinate stress-response pathways in the nucleus.
Mitochondria generate a major portion of ROS in roots but only a small portion
in leaves. While ETC complexes I–IV in plants are similar to those in animal
mitochondria, these complexes also contain five unique enzymes, including an
alternative oxidase (AOX) and four NAD(P)H dehydrogenases (Moller 2001; Jacoby
et al. 2012). Complex I and III are major sites for ROS production in plants. AOX
27
helps minimize ROS accumulation by the ETC (Moller 2001; Jacoby et al. 2012).
ABA and drought treatment promote ROS production in mitochondria (Rhoads et al.
2006; He et al. 2012; Yang et al. 2014). ABA OVERLY SENSITIVE6 (ABO6) encodes
a DEXH box RNA helicase that regulates the splicing of several genes in Complex I
(He et al. 2012). ABO8 encodes a pentatricopeptide repeat (PPR) protein involved in
mediating the splicing of NAD4 intron 3 in mitochondrial complex I (Yang et al.
2014). Both abo6 and abo8 mutants are hypersensitive to ABA in terms of seed
germination and primary root growth (He et al. 2012; Yang et al. 2014). Interestingly,
the expression of ABO6 and ABO8 is reduced by ABA treatment (He et al. 2012;
Yang et al. 2014). Both ABA treatment and drought stress promote greater ROS
accumulation in the abo6 and abo8 mutants compared to wild type (He et al. 2012;
Yang et al. 2014), indicating that ABA signaling regulates ROS production in
mitochondria. The abo8 mutant exhibits increased ROS accumulation in the root tip,
resulting in delayed differentiation of distal stem cells (DSC) (Yang et al. 2014).
Another study by Yu et al. showed that mutations in a P-loop NTPase localized to the
mitochondria in Arabidopsis reduce both H2O2 and O2.- levels in roots and promote
DSC differentiation (Yu et al. 2016). UPBEAT1 (UPB1) encodes a bHLH
transcriptional factor that negatively regulates the expression of several peroxidase
genes in the root elongation zone (Tsukagoshi et al. 2010). The upb1 mutant exhibits
increased O2.- levels in the root meristem and decrease apoplastic H2O2 content in the
elongation zone, which delays the transition from cell proliferation to differentiation,
resulting in enlarged root apical meristems (Tsukagoshi et al. 2010). ROS levels must
28
also be maintained at moderate levels in animal cells in order to maintain stem cell
homeostasis (Le Belle et al. 2011; Morimoto et al. 2013; Paul et al. 2014). These
studies indicate that ROS homeostasis is essential for cell division and differentiation
(Mittler 2017).
Peroxisomes mainly produce H2O2 and O2- through several metabolic pathways.
Water stress promotes stomatal closure and reduces CO2 assimilation, thus increasing
photorespiration and glycolate production. Glycolate from chloroplasts is oxidized by
glycolate oxidase (GOX) to produce glyoxylate and most of the H2O2 in leaf
peroxisomes, indicating that ROS signals are communicated between chloroplasts and
peroxisomes through various metabolites. Additionally, acyl-CoA oxidase (ACX)
catalyzes the production of H2O2 during peroxisomal fatty acid β-oxidation, a process
that occurs more actively in germinating seeds than in other tissues (Sandalio and
Romero-Puertas 2015). The matrix xanthine oxidase (XOD) in leaf peroxisomes
generates O2-, which is converted to H2O2 by SOD. H2O2 is mainly detoxified by
peroxisome-localized CATs in leaves (Miller et al. 2010). Furthermore, NO and its
derivative, RNS, are also produced in peroxisomes, which can influence many aspects
of cellular function in response to environmental stress (Sandalio and Romero-Puertas
2015).
ROS produced from different organelles may diffuse into the cytosol. Whether
these ROS have similar effects in the cytosol is not yet clear. However, ROS
produced from different organelles could form a specific signature to trigger unique
nuclear gene expression responses (Rosenwasser et al. 2011; Sewelam et al. 2014).
29
For example, chloroplastic H2O2 has a significant bias to induce the expression of
genes related to wounding and pathogen attack, while H2O2 from peroxisomes
regulates more genes involved in protein repair responses (Sewelam et al. 2014). As it
might not be possible for cellular components to determine which ROS comes from
which organelle, this finding suggests that ROS, with or without other components,
are involved in regulating the expression of specific genes.
Although ROS signals from different organelles can communicate with each
other, little is known about this communication process (Shapiguzov et al. 2012).
Mutations in Arabidopsis MOSAIC DEATH1 (MOD1), encoding an enoyl-acyl carrier
protein (ACP) reductase for fatty acid biosynthesis in chloroplasts, lead to high H2O2
and O2∙− production in mitochondria (Mou et al. 2000; Wu et al. 2015), suggesting
that chloroplast signals might be transmitted to mitochondria to modulate ROS
production. A screening for suppressors of mod1 identified several nuclear genes
encoding proteins involved in mitochondrial Complex I (Wu et al. 2015). In fact,
mutations in these of Complex I genes usually result in higher ROS levels than wild
type (Lee et al. 2002; Meyer et al. 2009; He et al. 2012). Signals from mod1
chloroplasts might enhance relative mitochondrial ETC activity, causing greater
electron leakage and more ROS to be produced in Complex I. This effect is
compromised by mutations in Complex I genes, as mod1 suppressor mutations reduce
ETC activity (Wu et al. 2015). The mod1 mutant and its suppressor lines represent
valuable materials for further studying ROS-related communication between
chloroplasts and mitochondria.
30
THE ROLE OF ROS SIGNALING IN CELL-TO-CELL AND
LONG-DISTANCE COMMUNICATION DURING STRESS
Besides inside cells, ROS communication occurs between cells. ROS, Ca2+ and
electric signals can be transmitted from the tissue of origin to distant tissues within
minutes, allowing plants to achieve systemic acquired acclimation (SAA) under
abiotic stress (Figure 5) (Mittler et al. 2011; Gilroy et al. 2016). Various abiotic
stresses, such as wounding, heat, cold, high intensity light and high salinity, trigger
the production of RBOHD-mediated apoplastic ROS, which move in relay-type
fashion in the apoplast from the local tissue throughout the entire plant at speeds of up
to 8.4 cm min–1 (Miller et al. 2009).
RBOHD-mediated ROS enter neighboring cells to trigger an increase in transient
Ca2+ levels. This, in turn, activates protein kinases such as CPK5 to phosphorylate and
activate RBOHD, thus forming a wave of ROS and Ca2+ that functions in cell-to-cell
and long-distance communication (Dubiella et al. 2013). Heat stress also transiently
increases the accumulation of ABA in systemic tissues in an RBOHD-dependent
manner. Blocking ABA biosynthesis or signaling impairs the SAA response to heat
stress, indicating the importance of coordination between ABA and the ROS wave in
SAA (Suzuki et al. 2013).
Osmotic stress quickly induces increases in [Ca2+]i levels through the action of
OSCA1 (REDUCED HYPEROSMOLARITY, INDUCED CA2+ INCREASE1), a
Ca2+ channel and osmosensor localized on the plasma membrane. However, while the
31
osca1 mutant shows a reduced Ca2+ response to osmotic stress, it still responds
normally to ABA and H2O2 treatment, suggesting that OSCA1 functions upstream of
ABA and H2O2 signaling (Yuan et al. 2014). Further studies are required to determine
whether OSCA1 is involved producing the Ca2+ wave.
The long distance, salt-stress-induced Ca2+ wave (which travels at a speed of 2.4
cm min–1) requires the vacuolar cation-permeable channel TWO PORE CHANNEL1
(TPC1) (Choi et al. 2014). TPC1 is sensitive to Ca2+ levels in both the cytosol and
vacuole lumen and is likely involved in calcium-induced calcium release during Ca2+
wave propagation (Choi et al. 2014). Glutamate receptor-like proteins (GLRs)
localized on the plasma membrane might also function as amino acid-gated
Ca²⁺channels (Kong et al. 2016). L-methionine (L-Met) activates GLR3.1 and
GLR3.5 Ca2+ channels to increase cytosolic Ca2+ levels, which is required for
NADPH oxidase-mediated ROS production and stomatal closure but functions
independently of ABA (Kong et al. 2016). Wounding rapidly induces distal systemic
electrical signals, a process that requires the participation of GLR3.2, GLR3.3 and
GLR3.6 (Mousavi et al. 2013). GLR3.3 and GLR3.6 are required for propagating
wound-induced electrical signals beyond the wounded leaf, while GLR3.5 is required
to prevent the formation of wound-induced electrical potentials in distal,
non-neighboring leaves (Salvador-Recatalà 2016). However, the molecular
connections among ROS/Ca2+ and electrical signals require further exploration.
FUTURE PROSPECTS
32
In this review, we summarized the molecular mechanisms that regulate the ROS
signaling network in plants, primarily under drought stress and pathogen attack.
Although we divided our review into several sections for the sake of discussion, ROS
production and signaling under different conditions cannot be separated within a cell.
Even under very strict experimental conditions, ROS production cannot be regulated
in only a specific site in the cell. Once a stress is applied, different types of ROS will
be produced immediately in different compartments and different sites of a cell. These
ROS form a complicated network, making it quite difficult to study ROS signaling.
Therefore, when investigating ROS signaling, we should take the whole picture into
account for their roles in plant responses to abiotic and biotic stress. While ROS are
metabolic by-products, high ROS levels are toxic to cells, and thus their
concentrations are kept low by the action of various antioxidants. At the same time,
ROS are absolutely required for many plant developmental processes and responses to
biotic and abiotic stress (Mittler 2017). For example, in rice dst (drought and salt
tolerance) mutant, it has shown that increasing ROS to an appropriate level does not
affect the plant growth and yield, but greatly increase plant tolerance to drought and
salt stress (Huang et al. 2009). DST encodes a zinc finger transcriptional factor that
likely directly regulates peroxidase 24 precursor for scavenging H2O2 (Huang et al.
2009). Wheat seedlings carrying a mutated Ta-sro1 gene encoding a poly(ADP ribose)
polymerase (PARP) domain protein with high PARP activity increase ROS
production and yield under salinity stress (Liu et al. 2014). These studies point out a
new strategy for breeding of stress resistant crops by modifying the ROS production
33
in the future. Although much exciting progress has been made, thanks largely to
newly developed techniques and rapid progress in genome and protein analysis
methods, a complete understanding of the exact nature of ROS signaling remains
elusive.
Some gaps in our knowledge are summarized by the following questions: How do
we measure the levels of different types of ROS and their dynamic changes at various
sites in the cell? How are ROS signals that are produced as a result of normal
developmental processes physically interconnected and integrated with those
produced as a result of biotic and abiotic stress? How are ROS differentially
recognized as second messengers in a cell or in neighboring cells during processes
such as PCD? Different ROS sensors must exist, but how do we identify these sensors?
Given the diversity of receptor-like protein kinases in plants (Shiu and Bleecker 2001),
do they directly sense and transmit apoplastic ROS signals? If so, how? Do they act
alone or together with other components to transduce a ROS wave? It is well known
that pretreatment with low concentration of exogenous H2O2 (called H2O2-priming)
can increase plant tolerance to various abiotic stresses (Hossain et al. 2015), but the
molecular mechanisms for this are not well understood. Right now, ROS level can be
controlled in crops by genetically manipulating the expression of ROS related genes.
But which ROS level should be maintained in order to increase stress tolerance
without apparent yield penalty needs further explored in various crops with different
genetic backgrounds.
Most studies on ROS tend to focus on their production and signal transduction in
34
the cytosol, organelles and plasma membrane. However, the effects of ROS on
chromatin structure and epigenetic modifications in plants require further exploration.
ACKNOWLEDGEMENTS
J.Z. and Z.G. are supported by the National Key Scientific Research Project
(2011CB915400). Z.G. is supported by the National Natural Science Foundation of
China (31730007).
35
REFERENCES
Arnaud D, Hwang I (2015) A sophisticated network of signaling pathways regulates stomatal defenses
to bacterial pathogens. Mol Plant 8: 566-581
Arnaud D, Lee S, Takebayashi Y, Choi D, Choi J, Sakakibara H, Hwang I (2017) Cytokinin-mediated
regulation of reactive oxygen species homeostasis modulates stomatal immunity in Arabidopsis. Plant
Cell 29: 543-559
Asgher M, Per TS, Masood A, Fatma M, Freschi L, Corpas FJ, Khan NA (2017) Nitric oxide signaling and
its crosstalk with other plant growth regulators in plant responses to abiotic stress. Environ Sci Pollut
Res 24: 2273-2285
Asselbergh B, Curvers K, Franca SC, Audenaert K, Vuylsteke M, Van Breusegem F, Hofte M (2007)
Resistance to Botrytis cinerea in sitiens, an abscisic acid-deficient tomato mutant, involves timely
production of hydrogen peroxide and cell wall modifications in the epidermis. Plant Physiol 144:
1863-1877
Baxter A, Mittler R, Suzuki N (2014) ROS as key players in plant stress signalling. J Exp Bot 65:
1229-1240
Bindschedler LV, Dewdney J, Blee KA, Stone JM, Asai T, Plotnikov J, Denoux C, Hayes T, Gerrish C,
Davies DR, Ausubel FM, Bolwell GP (2006) Peroxidase-dependent apoplastic oxidative burst in
Arabidopsis required for pathogen resistance. Plant J 47: 851-863
Bostock RM, Pye MF, Roubtsova TV (2014) Predisposition in plant disease: Exploiting the nexus in
abiotic and biotic stress perception and response. Annu Rev Phytopathol 52: 517-549
Boudsocq M, Willmann MR, McCormack M, Lee H, Shan LB, He P, Bush J, Cheng SH, Sheen J (2010)
Differential innate immune signalling via Ca2+ sensor protein kinases. Nature 464: 418-U116
Bourdais G, Burdiak P, Gauthier A, Nitsch L, Salojarvi J, Rayapuram C, Idanheimo N, Hunter K, Kimura S,
Merilo E, Vaattovaara A, Oracz K, Kaufholdt D, Pallon A, Anggoro DT, Glow D, Lowe J, Zhou J,
Mohammadi O, Puukko T, Albert A, Lang H, Ernst D, Kollist H, Brosche M, Durner J, Borst JW, Collinge
DB, Karpinski S, Lyngkjaer MF, Robatzek S, Wrzaczek M, Kangasjarvi J, Consortium C (2015) Large-scale
phenomics identifies primary and fine-tuning roles for CRKs in responses related to oxidative stress.
PLoS Genet 11:
Brandt B, Munemasa S, Wang C, Nguyen D, Yong TM, Yang PG, Poretsky E, Belknap TF, Waadt R,
Aleman F, Schroeder JI (2015) Calcium specificity signaling mechanisms in abscisic acid signal
transduction in Arabidopsis guard cells. eLife 4:
Bright J, Desikan R, Hancock JT, Weir IS, Neill SJ (2006) ABA-induced NO generation and stomatal
closure in Arabidopsis are dependent on H2O2 synthesis. Plant J 45: 113-122
Camejo D, Guzman-Cedeno A, Moreno A (2016) Reactive oxygen species, essential molecules, during
plant-pathogen interactions. Plant Physiol Biochem 103: 10-23
Chan KX, Mabbitt PD, Phua SY, Mueller JW, Nisar N, Gigolashvili T, Stroeher E, Grassl J, Arlt W,
Estavillo GM, Jackson CJ, Pogson BJ (2016) Sensing and signaling of oxidative stress in chloroplasts by
inactivation of the SAL1 phosphoadenosine phosphatase. Proc Natl Acad Sci USA 113: E4567-E4576
Chater C, Gray JE, Beerling DJ (2013) Early evolutionary acquisition of stomatal control and
development gene signalling networks. Curr Opin Plant Biol 16: 638-646
Chater C, Peng K, Movahedi M, Dunn JA, Walker HJ, Liang YK, McLachlan DH, Casson S, Isner JC,
36
Wilson I, Neill SJ, Hedrich R, Gray JE, Hetherington AM (2015) Elevated CO2-induced responses in
stomata require ABA and ABA signaling. Curr Biol 25: 2709-2716
Choi WG, Toyota M, Kim SH, Hilleary R, Gilroy S (2014) Salt stress-induced Ca2+ waves are associated
with rapid, long-distance root-to-shoot signaling in plants. Proc Natl Acad Sci USA 111: 6497-6502
Choudhury FK, Rivero RM, Blumwald E, Mittler R (2017) Reactive oxygen species, abiotic stress and
stress combination. Plant J 90: 856-867
Daudi A, Cheng ZY, O'Brien JA, Mammarella N, Khan S, Ausubel FM, Bolwell GP (2012) The apoplastic
oxidative burst peroxidase in Arabidopsis is a major component of pattern-triggered immunity. Plant
Cell 24: 275-287
de Torres-Zabala M, Truman W, Bennett MH, Lafforgue G, Mansfield JW, Egea PR, Bogre L, Grant M
(2007) Pseudomonas syringae pv. tomato hijacks the Arabidopsis abscisic acid signalling pathway to
cause disease. EMBO J 26: 1434-1443
Deger AG, Scherzer S, Nuhkat M, Kedzierska J, Kollist H, Brosche M, Unyayar S, Boudsocq M, Hedrich R,
Roelfsema MRG (2015) Guard cell SLAC1-type anion channels mediate flagellin-induced stomatal
closure. New Phytol 208: 162-173
Delledonne M, Zeier J, Marocco A, Lamb C (2001) Signal interactions between nitric oxide and reactive
oxygen intermediates in the plant hypersensitive disease resistance response. Proc Natl Acad Sci USA
98: 13454-13459
Dietz KJ, Turkan I, Krieger-Liszkay A (2016) Redox- and reactive oxygen species-dependent signaling
into and out of the photosynthesizing chloroplast. Plant Physiol 171: 1541-1550
Dou DL, Zhou JM (2012) Phytopathogen effectors subverting host immunity: Different foes, similar
battleground. Cell Host Microbe 12: 484-495
Drerup MM, Schlucking K, Hashimoto K, Manishankar P, Steinhorst L, Kuchitsu K, Kudla J (2013) The
calcineurin B-like calcium sensors CBL1 and CBL9 together with their interacting protein kinase CIPK26
regulate the Arabidopsis NADPH oxidase RBOHF. Mol Plant 6: 559-569
Dubiella U, Seybold H, Durian G, Komander E, Lassig R, Witte CP, Schulze WX, Romeis T (2013)
Calcium-dependent protein kinase/NADPH oxidase activation circuit is required for rapid defense
signal propagation. Proc Natl Acad Sci USA 110: 8744-8749
Engineer CB, Hashimoto-Sugimoto M, Negi J, Israelsson-Nordstrom M, Azoulay-Shemer T, Rappel WJ,
Iba K, Schroeder JI (2016) CO2 Sensing and CO2 regulation of stomatal conductance: Advances and
open questions. Trends Plant Sci 21: 16-30
Estavillo GM, Crisp PA, Pornsiriwong W, Wirtz M, Collinge D, Carrie C, Giraud E, Whelan J, David P,
Javot H, Brearley C, Hell R, Marin E, Pogson BJ (2011) Evidence for a SAL1-PAP Chloroplast retrograde
pathway that functions in drought and high light signaling in Arabidopsis. Plant Cell 23: 3992-4012
Fabre N, Reiter IM, Becuwe-Linka N, Genty B, Rumeau D (2007) Characterization and expression
analysis of genes encoding alpha and beta carbonic anhydrases in Arabidopsis. Plant Cell Environ 30:
617-629
Finkelstein RR, Gampala SSL, Rock CD (2002) Abscisic acid signaling in seeds and seedlings. Plant Cell
14: S15-S45
Galvez-Valdivieso G, Fryer MJ, Lawson T, Slattery K, Truman W, Smirnoff N, Asami T, Davies WJ, Jones
AM, Baker NR, Mullineaux PM (2009) The high light response in Arabidopsis involves ABA signaling
between vascular and bundle sheath cells. Plant Cell 21: 2143-2162
Geiger D, Maierhofer T, AL-Rasheid KAS, Scherzer S, Mumm P, Liese A, Ache P, Wellmann C, Marten I,
Grill E, Romeis T, Hedrich R (2011) Stomatal closure by fast abscisic acid signaling is mediated by the
37
guard cell anion channel SLAH3 and the receptor RCAR1. Sci Signal 4:
Geiger D, Scherzer S, Mumm P, Marten I, Ache P, Matschi S, Liese A, Wellmann C, Al-Rasheid KAS, Grill
E, Romeis T, Hedrich R (2010) Guard cell anion channel SLAC1 is regulated by CDPK protein kinases
with distinct Ca2+ affinities. Proc Natl Acad Sci USA 107: 8023-8028
Geiger D, Scherzer S, Mumm P, Stange A, Marten I, Bauer H, Ache P, Matschi S, Liese A, Al-Rasheid
KAS, Romeis T, Hedrich R (2009) Activity of guard cell anion channel SLAC1 is controlled by
drought-stress signaling kinase-phosphatase pair. Proc Natl Acad Sci USA 106: 21425-21430
Gilroy S, Bialasek M, Suzuki N, Gorecka M, Devireddy AR, Karpinski S, Mittler R (2016) ROS, calcium,
and electric signals: Key mediators of rapid systemic signaling in plants. Plant Physiol 171: 1606-1615
Grondin A, Rodrigues O, Verdoucq L, Merlot S, Leonhardt N, Maurel C (2015) Aquaporins contribute
to ABA-triggered stomatal closure through OST1-mediated phosphorylation. Plant Cell 27: 1945-1954
Hao HQ, Fan LS, Chen T, Li RL, Li XJ, He QH, Botella MA, Lin JX (2014) Clathrin and membrane
microdomains cooperatively regulate RbohD dynamics and activity in Arabidopsis. Plant Cell 26:
1729-1745
Hashimoto M, Negi J, Young J, Israelsson M, Schroeder JI, Iba K (2006) Arabidopsis HT1 kinase controls
stomatal movements in response to CO2. Nat Cell Biol 8: 391-U352
He JN, Duan Y, Hua DP, Fan GJ, Wang L, Liu Y, Chen ZZ, Han LH, Qu LJ, Gong ZZ (2012) DEXH box RNA
helicase-mediated mitochondrial reactive oxygen species production in Arabidopsis mediates
crosstalk between abscisic acid and auxin signaling. Plant Cell 24: 1815-1833
Horak H, Sierla M, Toldsepp K, Wang C, Wang YS, Nuhkat M, Valk E, Pechter P, Merilo E, Salojarvi J,
Overmyer K, Loog B, Brosche A, Schroeder JI, Kangasjarvi J, Kollist H (2016) A dominant mutation in
the HT1 kinase uncovers roles of MAP kinases and GHR1 in CO2-induced stomatal closure. Plant Cell
28: 2493-2509
Hossain MA, Bhattacharjee S, Armin SM, Qian PP, Xin W, Li HY, Burritt DJ, Fujita M, Tran LSP (2015)
Hydrogen peroxide priming modulates abiotic oxidative stress tolerance: Insights from ROS
detoxification and scavenging. Front Plant Sci 6:
Hu HH, Boisson-Dernier A, Israelsson-Nordstrom M, Bohmer M, Xue SW, Ries A, Godoski J, Kuhn JM,
Schroeder JI (2010) Carbonic anhydrases are upstream regulators of CO2-controlled stomatal
movements in guard cells. Nat Cell Biol 12: 87-U234
Hu JL, Huang XH, Chen LC, Sun XW, Lu CM, Zhang LX, Wang YC, Zuo JR (2015) Site-specific
nitrosoproteomic identification of endogenously S-nitrosylated proteins in Arabidopsis. Plant Physiol
167: 1731-1746
Hua DP, Wang C, He JN, Liao H, Duan Y, Zhu ZQ, Guo Y, Chen ZZ, Gong ZZ (2012) A plasma membrane
receptor kinase, GHR1, mediates abscisic acid- and hydrogen peroxide-regulated stomatal movement
in Arabidopsis. Plant Cell 24: 2546-2561
Huang XY, Chao DY, Gao JP, Zhu MZ, Shi M, Lin HX (2009) A previously unknown zinc finger protein,
DST, regulates drought and salt tolerance in rice via stomatal aperture control. Genes Dev 23:
1805-1817
Idanheimo N, Gauthier A, Salojarvi J, Siligato R, Brosche M, Kollist H, Mahonen AP, Kangasjarvi J,
Wrzaczek M (2014) The Arabidopsis thaliana cysteine-rich receptor-like kinases CRK6 and CRK7
protect against apoplastic oxidative stress. Biochem Biophys Res Commun 445: 457-462
Imes D, Mumm P, Bohm J, Al-Rasheid KAS, Marten I, Geiger D, Hedrich R (2013) Open stomata 1
(OST1) kinase controls R-type anion channel QUAC1 in Arabidopsis guard cells. Plant J 74: 372-382
Jacoby RP, Li L, Huang SB, Lee C, Millar AH, Taylor NL (2012) Mitochondrial composition, function and
38
stress response in plants. J Integr Plant Biol 54: 887-906
Jakobson L, Vaahtera L, Toldsepp K, Nuhkat M, Wang C, Wang YS, Horak H, Valk E, Pechter P,
Sindarovska Y, Tang J, Xiao CL, Xu Y, Talas UG, Garcia-Sosa AT, Kangasjarvi S, Maran U, Remm M,
Roelfsema MRG, Hu HH, Kangasjarvi J, Loog M, Schroeder JI, Kollist H, Brosche M (2016) Natural
variation in Arabidopsis Cvi-0 accession reveals an important role of MPK12 in guard cell CO2 signaling.
PLoS Biol 14:
Jammes F, Song C, Shin DJ, Munemasa S, Takeda K, Gu D, Cho D, Lee S, Giordo R, Sritubtim S,
Leonhardt N, Ellis BE, Murata Y, Kwak JM (2009) MAP kinases MPK9 and MPK12 are preferentially
expressed in guard cells and positively regulate ROS-mediated ABA signaling. Proc Natl Acad Sci USA
106: 20520-20525
Kadota Y, Shirasu K, Zipfel C (2015) Regulation of the NADPH Oxidase RBOHD during plant immunity.
Plant Cell Physiol 56: 1472-1480
Kadota Y, Sklenar J, Derbyshire P, Stransfeld L, Asai S, Ntoukakis V, Jones JDG, Shirasu K, Menke F,
Jones A, Zipfel C (2014) Direct Regulation of the NADPH Oxidase RBOHD by the PRR-associated kinase
BIK1 during plant immunity. Mol Cell 54: 43-55
Kaliff M, Staal J, Myrenas M, Dixelius C (2007) ABA is required for Leptosphaeria maculans resistance
via ABI1- and ABI4-dependent signaling. Mol Plant Microbe Interact 20: 335-345
Kim TH, Bohmer M, Hu HH, Nishimura N, Schroeder JI (2010) Guard cell signal transduction network:
Advances in understanding abscisic acid, CO2, and Ca2+ signaling. Annu Rev Plant Biol 61: 561-591
Kocsy G, Tari I, Vankova R, Zechmann B, Gulyas Z, Poor P, Galiba G (2013) Redox control of plant
growth and development. Plant Sci 211: 77-91
Kolla VA, Vavasseur A, Raghavendra AS (2007) Hydrogen peroxide production is an early event during
bicarbonate induced stomatal closure in abaxial epidermis of Arabidopsis. Planta 225: 1421-1429
Kong D, Hu HC, Okuma E, Lee Y, Lee HS, Munemasa S, Cho D, Ju C, Pedoeim L, Rodriguez B, Wang J, Im
W, Murata Y, Pei ZM, Kwak JM (2016) L-met activates Arabidopsis GLR Ca2+ channels upstream of ROS
production and regulates stomatal movement. Cell Rep 17: 2553-2561
Kong L, Cheng J, Zhu Y, Ding Y, Meng J, Chen Z, Xie Q, Guo Y, Li J, Yang S, Gong Z (2015) Degradation of
the ABA co-receptor ABI1 by PUB12/13 U-box E3 ligases. Nat Commun 6: 8630
Kwak JM, Mori IC, Pei ZM, Leonhardt N, Torres MA, Dangl JL, Bloom RE, Bodde S, Jones JDG,
Schroeder JI (2003) NADPH oxidase AtrbohD and AtrbohF genes function in ROS-dependent ABA
signaling in Arabidopsis. EMBO J 22: 2623-2633
Laloi C, Stachowiak M, Pers-Kamczyc E, Warzych E, Murgia I, Apel K (2007) Cross-talk between singlet
oxygen- and hydrogen peroxide-dependent signaling of stress responses in Arabidopsis thaliana. Proc
Natl Acad Sci USA 104: 672-677
Le Belle JE, Orozco NM, Paucar AA, Saxe JP, Mottahedeh J, Pyle AD, Wu H, Kornblum HI (2011)
Proliferative neural stem cells have high endogenous ROS levels that regulate self-renewal and
neurogenesis in a PI3K/Akt-dependant manner. Cell Stem Cell 8: 59-71
Lee BH, Lee HJ, Xiong LM, Zhu JK (2002) A mitochondrial complex I defect impairs cold-regulated
nuclear gene expression. Plant Cell 14: 1235-1251
Lee KP, Kim C, Landgraf F, Apel K (2007) EXECUTER1- and EXECUTER2-dependent transfer of
stress-related signals from the plastid to the nucleus of Arabidopsis thaliana. Proc Natl Acad Sci USA
104: 10270-10275
Li L, Li M, Yu LP, Zhou ZY, Liang XX, Liu ZX, Cai GH, Gao LY, Zhang XJ, Wang YC, Chen S, Zhou JM (2014)
The FLS2-associated kinase BIK1 directly phosphorylates the NADPH oxidase RbohD to control plant
39
immunity. Cell Host Microbe 15: 329-338
Liang XX, Ding PT, Liang KH, Wang JL, Ma MM, Li L, Li L, Li M, Zhang XJ, Chen S, Zhang YL, Zhou JM
(2016) Arabidopsis heterotrimeric G proteins regulate immunity by directly coupling to the FLS2
receptor. eLife 5:
Lind C, Dreyer I, Lopez-Sanjurjo EJ, von Meyer K, Ishizaki K, Kohchi T, Lang D, Zhao Y, Kreuzer I,
Al-Rasheid KAS, Ronne H, Reski R, Zhu JK, Geiger D, Hedrich R (2015) Stomatal guard cells co-opted an
ancient ABA-dependent desiccation survival system to regulate stomatal closure. Curr Biol 25:
928-935
Liu ST, Liu SW, Wang M, Wei TD, Meng C, Wang M, Xia GM (2014) A wheat SIMILAR TO RCD-ONE gene
enhances seedling growth and abiotic stress resistance by modulating redox homeostasis and
maintaining genomic integrity. Plant Cell 26: 164-180
Liu Y, He JN, Chen ZZ, Ren XZ, Hong XH, Gong ZZ (2010) ABA overly-sensitive 5 (ABO5), encoding a
pentatricopeptide repeat protein required for cis-splicing of mitochondrial nad2 intron 3, is involved
in the abscisic acid response in Arabidopsis. Plant J 63: 749-765
Liu ZX, Wu Y, Yang F, Zhang YY, Chen S, Xie Q, Tian XJ, Zhou JM (2013) BIK1 interacts with PEPRs to
mediate ethylene-induced immunity. Proc Natl Acad Sci USA 110: 6205-6210
Lu DP, Wu SJ, Gao XQ, Zhang YL, Shan LB, He P (2010) A receptor-like cytoplasmic kinase, BIK1,
associates with a flagellin receptor complex to initiate plant innate immunity. Proc Natl Acad Sci USA
107: 496-501
Ma Y, Szostkiewicz I, Korte A, Moes D, Yang Y, Christmann A, Grill E (2009) Regulators of PP2C
phosphatase activity function as abscisic acid sensors. Science 324: 1064-1068
Medlyn BE, Barton CVM, Broadmeadow MSJ, Ceulemans R, De Angelis P, Forstreuter M, Freeman M,
Jackson SB, Kellomaki S, Laitat E, Rey A, Roberntz P, Sigurdsson BD, Strassemeyer J, Wang K, Curtis PS,
Jarvis PG (2001) Stomatal conductance of forest species after long-term exposure to elevated CO2
concentration: A synthesis. New Phytol 149: 247-264
Meinhard M, Grill E (2001) Hydrogen peroxide is a regulator of ABI1, a protein phosphatase 2C from
Arabidopsis. FEBS Lett 508: 443-446
Meinhard M, Rodriguez PL, Grill E (2002) The sensitivity of ABI2 to hydrogen peroxide links the
abscisic acid-response regulator to redox signalling. Planta 214: 775-782
Melotto M, Underwood W, He SY (2008) Role of stomata in plant innate immunity and foliar bacterial
diseases. Annu Rev Phytopathol 46: 101-122
Melotto M, Underwood W, Koczan J, Nomura K, He SY (2006) Plant stomata function in innate
immunity against bacterial invasion. Cell 126: 969-980
Meng XZ, Xu J, He YX, Yang KY, Mordorski B, Liu YD, Zhang SQ (2013) Phosphorylation of an ERF
Transcription Factor by Arabidopsis MPK3/MPK6 regulates plant defense gene induction and fungal
resistance. Plant Cell 25: 1126-1142
Meng XZ, Zhang SQ (2013) MAPK cascades in plant disease resistance signaling. Annu Rev
Phytopathol 51: 245-266
Merilo E, Laanemets K, Hu HH, Xue SW, Jakobson L, Tulva I, Gonzalez-Guzman M, Rodriguez PL,
Schroeder JI, Brosche M, Kollist H (2013) PYR/RCAR receptors contribute to ozone-, reduced air
humidity-, darkness-, and CO2-induced stomatal regulation. Plant Physiol 162: 1652-1668
Meskauskiene R, Nater M, Goslings D, Kessler F, den Camp RO, Apel K (2001) FLU: A negative
regulator of chlorophyll biosynthesis in Arabidopsis thaliana. Proc Natl Acad Sci USA 98: 12826-12831
Meyer EH, Tomaz T, Carroll AJ, Estavillo G, Delannoy E, Tanz SK, Small ID, Pogson BJ, Millar AH (2009)
40
Remodeled respiration in ndufs4 with low phosphorylation efficiency suppresses Arabidopsis
germination and growth and alters control of metabolism at night. Plant Physiol 151: 603-619
Meyer S, Mumm P, Imes D, Endler A, Weder B, Al-Rasheid KAS, Geiger D, Marten I, Martinoia E,
Hedrich R (2010) AtALMT12 represents an R-type anion channel required for stomatal movement in
Arabidopsis guard cells. Plant J 63: 1054-1062
Miao YC, Song CP, Dong FC, XC. W (2000) ABA-induced hydrogen peroxide generation in guard cells of
Vicia faba. Acta Phytophysiol Sin ( In Chinese) 26: 53-58
Miao YC, Lv D, Wang PC, Wang XC, Chen J, Miao C, Song CP (2006) An Arabidopsis glutathione
peroxidase functions as both a redox transducer and a scavenger in abscisic acid and drought stress
responses. Plant Cell 18: 2749-2766
Miller G, Schlauch K, Tam R, Cortes D, Torres MA, Shulaev V, Dangl JL, Mittler R (2009) The plant
NADPH oxidase RBOHD mediates rapid systemic signaling in response to diverse stimuli. Sci Signal 2:
Miller G, Suzuki N, Ciftci-Yilmaz S, Mittler R (2010) Reactive oxygen species homeostasis and signalling
during drought and salinity stresses. Plant Cell Environ 33: 453-467
Mittler R (2017) ROS are good. Trends Plant Sci 22: 11-19
Mittler R, Vanderauwera S, Gollery M, Van Breusegem F (2004) Reactive oxygen gene network of
plants. Trends Plant Sci 9: 490-498
Mittler R, Vanderauwera S, Suzuki N, Miller G, Tognetti VB, Vandepoele K, Gollery M, Shulaev V, Van
Breusegem F (2011) ROS signaling: the new wave? Trends Plant Sci 16: 300-309
Moller IM (2001) PLANT MITOCHONDRIA AND OXIDATIVE STRESS: Electron transport, NADPH
turnover, and metabolism of reactive oxygen species. Annu Rev Plant Physiol Plant Mol Biol 52:
561-591
Monaghan J, Matschi S, Shorinola O, Rovenich H, Matei A, Segonzac C, Malinovsky FG, Rathjen JP,
MacLean D, Romeis T, Zipfel C (2014) The calcium-dependent protein kinase CPK28 buffers plant
immunity and regulates BIK1 turnover. Cell Host Microbe 16: 605-615
Morimoto H, Iwata K, Ogonuki N, Inoue K, Atsuo O, Kanatsu-Shinohara M, Morimoto T,
Yabe-Nishimura C, Shinohara T (2013) ROS are required for mouse spermatogonial stem cell
self-renewal. Cell Stem Cell 12: 774-786
Mou ZL, He YK, Dai Y, Liu XF, Li JY (2000) Deficiency in fatty acid synthase leads to premature cell
death and dramatic alterations in plant morphology. Plant Cell 12: 405-417
Mousavi SAR, Chauvin A, Pascaud F, Kellenberger S, Farmer EE (2013) GLUTAMATE RECEPTOR-LIKE
genes mediate leaf-to-leaf wound signalling. Nature 500: 422-+
Murata Y, Pei ZM, Mori IC, Schroeder J (2001) Abscisic acid activation of plasma membrane Ca2+
channels in guard cells requires cytosolic NAD(P)H and is differentially disrupted upstream and
downstream of reactive oxygen species production in abi1-1 and abi2-1 protein phosphatase 2C
mutants. Plant Cell 13: 2513-2523
Mustilli AC, Merlot S, Vavasseur A, Fenzi F, Giraudat J (2002) Arabidopsis OST1 protein kinase
mediates the regulation of stomatal aperture by abscisic acid and acts upstream of reactive oxygen
species production. Plant Cell 14: 3089-3099
Negi J, Matsuda O, Nagasawa T, Oba Y, Takahashi H, Kawai-Yamada M, Uchimiya H, Hashimoto M, Iba
K (2008) CO2 regulator SLAC1 and its homologues are essential for anion homeostasis in plant cells.
Nature 452: 483-486
Neill S, Desikan R, Hancock J (2002) Hydrogen peroxide signalling. Curr Opin Plant Biol 5: 388-395
Nomura H, Komori T, Uemura S, Kanda Y, Shimotani K, Nakai K, Furuichi T, Takebayashi K, Sugimoto T,
41
Sano S, Suwastika IN, Fukusaki E, Yoshioka H, Nakahira Y, Shiina T (2012) Chloroplast-mediated
activation of plant immune signalling in Arabidopsis. Nat Commun 3:
O'Brien JA, Daudi A, Butt VS, Bolwell GP (2012) Reactive oxygen species and their role in plant defence
and cell wall metabolism. Planta 236: 765-779
Park SY, Fung P, Nishimura N, Jensen DR, Fujii H, Zhao Y, Lumba S, Santiago J, Rodrigues A, Chow TFF,
Alfred SE, Bonetta D, Finkelstein R, Provart NJ, Desveaux D, Rodriguez PL, McCourt P, Zhu JK,
Schroeder JI, Volkman BF, Cutler SR (2009) Abscisic acid inhibits type 2C protein phosphatases via the
PYR/PYL family of START proteins. Science 324: 1068-1071
Paul MK, Bisht B, Darmawan DO, Chiou R, Ha VL, Wallace WD, Chon AT, Hegab AE, Grogan T, Elashoff
DA, Alva-Ornelas JA, Gomperts BN (2014) Dynamic changes in intracellular ROS levels regulate airway
basal stem cell homeostasis through Nrf2-dependent notch signaling. Cell Stem Cell 15: 199-214
Pei ZM, Murata Y, Benning G, Thomine S, Klusener B, Allen GJ, Grill E, Schroeder JI (2000) Calcium
channels activated by hydrogen peroxide mediate abscisic acid signalling in guard cells. Nature 406:
731-734
Petersen LN, Ingle RA, Knight MR, Denby KJ (2009) OXI1 protein kinase is required for plant immunity
against Pseudomonas syringae in Arabidopsis. J Exp Bot 60: 3727-3735
Petrov V, Hille J, Mueller-Roeber B, Gechev TS (2015) ROS-mediated abiotic stress-induced
programmed cell death in plants. Front Plant Sci 6:
Pornsiriwong W, Estavillo GM, Chan KX, Tee EE, Ganguly D, Crisp PA, Phua SY, Zhao C, Qiu J, Park J,
Yong MT, Nisar N, Yadav AK, Schwessinger B, Rathjen J, Cazzonelli CI, Wilson PB, Gilliham M, Chen ZH,
Pogson BJ (2017) A chloroplast retrograde signal, 3'-phosphoadenosine 5'-phosphate, acts as a
secondary messenger in abscisic acid signaling in stomatal closure and germination. eLife 6:
Qi J, Wang J, Gong Z, Zhou JM (2017) Apoplastic ROS signaling in plant immunity. Curr Opin Plant Biol
38: 92-100
Qiao WH, Fan LM (2008) Nitric oxide signaling in plant responses to abiotic stresses. J Integr Plant Biol
50: 1238-1246
Ramel F, Birtic S, Ginies C, Soubigou-Taconnat L, Triantaphylides C, Havaux M (2012) Carotenoid
oxidation products are stress signals that mediate gene responses to singlet oxygen in plants. Proc
Natl Acad Sci USA 109: 5535-5540
Ramel F, Ksas B, Akkari E, Mialoundama AS, Monnet F, Krieger-Liszkay A, Ravanat JL, Mueller MJ,
Bouvier F, Havaux M (2013) Light-induced acclimation of the Arabidopsis chlorina1 mutant to singlet
oxygen. Plant Cell 25: 1445-1462
Rentel MC, Lecourieux D, Ouaked F, Usher SL, Petersen L, Okamoto H, Knight H, Peck SC, Grierson CS,
Hirt H, Knight MR (2004) OXI1 kinase is necessary for oxidative burst-mediated signalling in
Arabidopsis. Nature 427: 858-861
Rhoads DM, Umbach AL, Subbaiah CC, Siedow JN (2006) Mitochondrial reactive oxygen species.
Contribution to oxidative stress and interorganellar signaling. Plant Physiol 141: 357-366
Rodrigues O, Reshetnyak G, Grondin A, Saijo Y, Leonhardt N, Maurel C, Verdoucq L (2017) Aquaporins
facilitate hydrogen peroxide entry into guard cells to mediate ABA- and pathogen-triggered stomatal
closure. Proc Natl Acad Sci USA 114: 9200-9205
Rodriguez-Herva JJ, Gonzalez-Melendi P, Cuartas-Lanza R, Antunez-Lamas M, Rio-Alvarez I, Li Z,
Lopez-Torrejon G, Diaz I, del Pozo JC, Chakravarthy S, Collmer A, Rodriguez-Palenzuela P,
Lopez-Solanilla E (2012) A bacterial cysteine protease effector protein interferes with photosynthesis
to suppress plant innate immune responses. Cell Microbiol 14: 669-681
42
Rosenwasser S, Rot I, Sollner E, Meyer AJ, Smith Y, Leviatan N, Fluhr R, Friedman H (2011) Organelles
contribute differentially to reactive oxygen species-related events during extended darkness. Plant
Physiol 156: 185-201
Salvador-Recatalà V (2016) New roles for the GLUTAMATE RECEPTOR-LIKE 3.3, 3.5, and 3.6 genes as
on/off switches of wound-induced systemic electrical signals. Plant Signal Behav 11: e1161879
Sandalio LM, Romero-Puertas MC (2015) Peroxisomes sense and respond to environmental cues by
regulating ROS and RNS signalling networks. Ann Bot 116: 475-485
Sang YM, Cui DC, Wang XM (2001a) Phospholipase D and phosphatidic acid-mediated generation of
superoxide in arabidopsis. Plant Physiol 126: 1449-1458
Sang YM, Zheng SQ, Li WQ, Huang BR, Wang XM (2001b) Regulation of plant water loss by
manipulating the expression of phospholipase D alpha. Plant J 28: 135-144
Sato A, Sato Y, Fukao Y, Fujiwara M, Umezawa T, Shinozaki K, Hibi T, Taniguchi M, Miyake H, Goto DB,
Uozumi N (2009) Threonine at position 306 of the KAT1 potassium channel is essential for channel
activity and is a target site for ABA-activated SnRK2/OST1/SnRK2.6 protein kinase. Biochem J 424:
439-448
Schroeder JI, Kwak JM, Allen GJ (2001) Guard cell abscisic acid signalling and engineering drought
hardiness in plants. Nature 410: 327-330
Sewelam N, Jaspert N, Van der Kelen K, Tognetti VB, Schmitz J, Frerigmann H, Stahl E, Zeier J, Van
Breusegem F, Maurino VG (2014) Spatial H2O2 signaling specificity: H2O2 from chloroplasts and
peroxisomes modulates the plant transcriptome differentially. Mol Plant 7: 1191-1210
Shang Y, Dai C, Lee MM, Kwak JM, Nam KH (2016) BRI1-associated receptor kinase 1 regulates guard
cell ABA signaling mediated by open stomata 1 in Arabidopsis. Mol Plant 9: 447-460
Shapiguzov A, Vainonen JP, Wrzaczek M, Kangasjarvi J (2012) ROS-talk - how the apoplast, the
chloroplast, and the nucleus get the message through. Front Plant Sci 3:
Shi H, Shen QJ, Qi YP, Yan HJ, Nie HZ, Chen YF, Zhao T, Katagiri F, Tang DZ (2013) BR-SIGNALING
KINASE1 physically associates with FLAGELLIN SENSING2 and regulates plant innate immunity in
Arabidopsis. Plant Cell 25: 1143-1157
Shi K, Li X, Zhang H, Zhang GQ, Liu YR, Zhou YH, Xia XJ, Chen ZX, Yu JQ (2015) Guard cell hydrogen
peroxide and nitric oxide mediate elevated CO2-induced stomatal movement in tomato. New Phytol
208: 342-353
Shiu SH, Bleecker AB (2001) Plant receptor-like kinase gene family: Diversity, function, and signaling.
Sci STKE 2001: re22
Shumbe L, Chevalier A, Legeret B, Taconnat L, Monnet F, Havaux M (2016) Singlet oxygen-induced cell
death in Arabidopsis under high-light stress is controlled by OXI1 kinase. Plant Physiol 170: 1757-1771
Sierla M, Waszczak C, Vahisalu T, Kangasjarvi J (2016) Reactive oxygen species in the regulation of
stomatal movements. Plant Physiol 171: 1569-1580
Singh R, Singh S, Parihar P, Mishra RK, Tripathi DK, Singh VP, Chauhan DK, Prasad SM (2016) Reactive
oxygen species (ROS): Beneficial companions of plants' developmental processes. Front Plant Sci 7:
1-19
Sirichandra C, Gu D, Hu HC, Davanture M, Lee S, Djaoui M, Valot B, Zivy M, Leung J, Merlot S, Kwak JM
(2009) Phosphorylation of the Arabidopsis AtrbohF NADPH oxidase by OST1 protein kinase. FEBS Lett
583: 2982-2986
Sussmilch FC, Brodribb TJ, McAdam SAM (2017) What are the evolutionary origins of stomatal
responses to abscisic acid in land plants? J Integr Plant Biol 59: 240-260
43
Suzuki N, Koussevitzky S, Mittler R, Miller G (2012) ROS and redox signalling in the response of plants
to abiotic stress. Plant Cell Environ 35: 259-270
Suzuki N, Miller G, Morales J, Shulaev V, Torres MA, Mittler R (2011) Respiratory burst oxidases: The
engines of ROS signaling. Curr Opin Plant Biol 14: 691-699
Suzuki N, Miller G, Salazar C, Mondal HA, Shulaev E, Cortes DF, Shuman JL, Luo XZ, Shah J, Schlauch K,
Shulaev V, Mittler R (2013) Temporal-spatial interaction between reactive oxygen species and abscisic
acid regulates rapid systemic acclimation in plants. Plant Cell 25: 3553-3569
Tang WQ, Kim TW, Oses-Prieto JA, Sun Y, Deng ZP, Zhu SW, Wang RJ, Burlingame AL, Wang ZY (2008)
BSKs mediate signal transduction from the receptor kinase BRI1 in Arabidopsis. Science 321: 557-560
Tian S, Wang XB, Li P, Wang H, Ji HT, Xie JY, Qiu QL, Shen D, Dong HS (2016) Plant aquaporin AtPIP1;4
links apoplastic H2O2 induction to disease immunity pathways. Plant Physiol 171: 1635-1650
Ton J, Flors V, Mauch-Mani B (2009) The multifaceted role of ABA in disease resistance. Trends Plant
Sci 14: 310-317
Torres MA, Dangl JL, Jones JDG (2002) Arabidopsis gp91(phox) homologues AtrbohD and AtrbohF are
required for accumulation of reactive oxygen intermediates in the plant defense response. Proc Natl
Acad Sci USA 99: 517-522
Triantaphylides C, Havaux M (2009) Singlet oxygen in plants: Production, detoxification and signaling.
Trends Plant Sci 14: 219-228
Tsukagoshi H, Busch W, Benfey PN (2010) Transcriptional regulation of ROS controls transition from
proliferation to differentiation in the root. Cell 143: 606-616
Umezawa T, Nakashima K, Miyakawa T, Kuromori T, Tanokura M, Shinozaki K, Yamaguchi-Shinozaki K
(2010) Molecular basis of the core regulatory network in ABA responses: Sensing, signaling and
transport. Plant Cell Physiol 51: 1821-1839
Vahisalu T, Kollist H, Wang YF, Nishimura N, Chan WY, Valerio G, Lamminmaki A, Brosche M, Moldau
H, Desikan R, Schroeder JI, Kangasjarvi J (2008) SLAC1 is required for plant guard cell S-type anion
channel function in stomatal signalling. Nature 452: 487-U415
Vogel MO, Moore M, Konig K, Pecher P, Alsharafa K, Lee J, Dietz KJ (2014) Fast retrograde signaling in
response to high light involves metabolite export, MITOGEN-ACTIVATED PROTEIN KINASE6, and
AP2/ERF transcription factors in Arabidopsis. Plant Cell 26: 1151-1165
Wagner D, Przybyla D, Camp ROD, Kim C, Landgraf F, Lee KP, Wursch M, Laloi C, Nater M, Hideg E,
Apel K (2004) The genetic basis of singlet oxygen-induced stress responses of Arabidopsis thaliana.
Science 306: 1183-1185
Wang C, Hu HH, Qin X, Zeise B, Xu DY, Rappel WJ, Boron WF, Schroeder JI (2016) Reconstitution of
CO2 regulation of SLAC1 anion channel and function of CO2-permeable PIP2;1 aquaporin as CARBONIC
ANHYDRASE4 interactor. Plant Cell 28: 568-582
Wang PC, Du YY, Li YA, Ren DT, Song CP (2010) Hydrogen peroxide-mediated activation of MAP kinase
6 modulates nitric oxide biosynthesis and signal transduction in Arabidopsis. Plant Cell 22: 2981-2998
Wang PC, Du YY, Zhao XL, Miao YC, Song CP (2013a) The MPK6-ERF6-ROS-responsive cis-acting
element7/GCC box complex modulates oxidative gene transcription and the oxidative response in
Arabidopsis. Plant Physiol 161: 1392-1408
Wang PC, Dua YY, Hou YJ, Zhao Y, Hsu CC, Yuan FJ, Zhu XH, Tao WA, Song CP, Zhu JK (2015a) Nitric
oxide negatively regulates abscisic acid signaling in guard cells by S-nitrosylation of OST1. Proc Natl
Acad Sci USA 112: 613-618
Wang PC, Xue L, Batelli G, Lee S, Hou YJ, Van Oosten MJ, Zhang HM, Tao WA, Zhu JK (2013b)
44
Quantitative phosphoproteomics identifies SnRK2 protein kinase substrates and reveals the effectors
of abscisic acid action. Proc Natl Acad Sci USA 110: 11205-11210
Wang ZP, Xing HL, Dong L, Zhang HY, Han CY, Wang XC, Chen QJ (2015b) Egg cell-specific
promoter-controlled CRISPR/Cas9 efficiently generates homozygous mutants for multiple target
genes in Arabidopsis in a single generation. Genome Biol 16:
Webb AAR, Hetherington AM (1997) Convergence of the abscisic acid, CO2, and extracellular calcium
signal transduction pathways in stomatal guard cells. Plant Physiol 114: 1557-1560
Wrzaczek M, Brosche M, Kangasjarvi J (2013) ROS signaling loops - production, perception, regulation.
Curr Opin Plant Biol 16: 575-582
Wu J, Sun YF, Zhao YN, Zhang J, Luo LL, Li M, Wang JL, Yu H, Liu GF, Yang LS, Xiong GS, Zhou JM, Zuo JR,
Wang YH, Li JY (2015) Deficient plastidic fatty acid synthesis triggers cell death by modulating
mitochondrial reactive oxygen species. Cell Res 25: 621-633
Wu L, Chen H, Curtis C, Fu ZQ (2014) Go in for the kill How plants deploy effector-triggered immunity
to combat pathogens. Virulence 5: 710-721
Xin XF, Nomura K, Aung K, Velasquez AC, Yao J, Boutrot F, Chang JH, Zipfel C, He SY (2016) Bacteria
establish an aqueous living space in plants crucial for virulence. Nature 539: 524-+
Xu ZZ, Jiang YL, Zhou GS (2015) Response and adaptation of photosynthesis, respiration, and
antioxidant systems to elevated CO2 with environmental stress in plants. Front Plant Sci 6:
Xue SW, Hu HH, Ries A, Merilo E, Kollist H, Schroeder JI (2011) Central functions of bicarbonate in
S-type anion channel activation and OST1 protein kinase in CO2 signal transduction in guard cell.
EMBO J 30: 1645-1658
Yamamoto Y, Negi J, Wang C, Isogai Y, Schroeder JI, Iba K (2016) The transmembrane region of guard
cell SLAC1 channels perceives CO2 signals via an ABA-independent pathway in Arabidopsis. Plant Cell
28: 557-567
Yang HJ, Mu JY, Chen LC, Feng J, Hu JL, Li L, Zhou JM, Zuo JR (2015) S-nitrosylation positively regulates
ascorbate peroxidase activity during plant stress responses. Plant Physiol 167: 1604-U1753
Yang L, Zhang J, He JN, Qin YY, Hua DP, Duan Y, Chen ZZ, Gong ZZ (2014) ABA-mediated ROS in
mitochondria regulate root meristem activity by controlling PLETHORA expression in Arabidopsis.
PLoS Genet 10:
Yoshida R, Umezawa T, Mizoguchi T, Takahashi S, Takahashi F, Shinozaki K (2006) The regulatory
domain of SRK2E/OST1/SnRK2.6 interacts with ABI1 and integrates abscisic acid (ABA) and osmotic
stress signals controlling stomatal closure in Arabidopsis. J Biol Chem 281: 5310-5318
Young JJ, Mehta S, Israelsson M, Godoski J, Grill E, Schroeder JI (2006) CO2 signaling in guard cells:
Calcium sensitivity response modulation, a Ca2+-independent phase, and CO2 insensitivity of the gca2
mutant. Proc Natl Acad Sci USA 103: 7506-7511
Yu QQ, Tian HY, Yue K, Liu JJ, Zhang B, Li XG, Ding ZJ (2016) A P-loop NTPase regulates quiescent
center cell division and distal stem cell identity through the regulation of ROS homeostasis in
Arabidopsis root. PLoS Genet 12:
Yuan F, Yang HM, Xue Y, Kong DD, Ye R, Li CJ, Zhang JY, Theprungsirikul L, Shrift T, Krichilsky B,
Johnson DM, Swift GB, He YK, Siedow JN, Pei ZM (2014) OSCA1 mediates osmotic-stress-evoked Ca2+
increases vital for osmosensing in Arabidopsis. Nature 514: 367
Yun BW, Feechan A, Yin MH, Saidi NBB, Le Bihan T, Yu M, Moore JW, Kang JG, Kwon E, Spoel SH, Pallas
JA, Loake GJ (2011) S-nitrosylation of NADPH oxidase regulates cell death in plant immunity. Nature
478: 264-U161
45
Zabala MDT, Littlejohn G, Jayaraman S, Studholme D, Bailey T, Lawson T, Tillich M, Licht D, Bolter B,
Delfino L, Truman W, Mansfield J, Smirnoff N, Grant M (2015) Chloroplasts play a central role in plant
defence and are targeted by pathogen effectors. Nat Plant 1:
Zhang J, Li W, Xiang TT, Liu ZX, Laluk K, Ding XJ, Zou Y, Gao MH, Zhang XJ, Chen S, Mengiste T, Zhang
YL, Zhou JM (2010) Receptor-like cytoplasmic kinases integrate signaling from multiple plant immune
receptors and are targeted by a Pseudomonas syringae effector. Cell Host Microbe 7: 290-301
Zhang W, Jeon BW, Assmann SM (2011) Heterotrimeric G-protein regulation of ROS signalling and
calcium currents in Arabidopsis guard cells. J Exp Bot 62: 2371-2379
Zhang WH, Qin CB, Zhao J, Wang XM (2004) Phospholipase D alpha 1-derived phosphatidic acid
interacts with ABI1 phosphatase 2C and regulates abscisic acid signaling. Proc Natl Acad Sci USA 101:
9508-9513
Zhang YY, Zhu HY, Zhang Q, Li MY, Yan M, Wang R, Wang LL, Welti R, Zhang WH, Wang XM (2009)
Phospholipase D alpha 1 and phosphatidic acid regulate NADPH oxidase activity and production of
reactive oxygen species in ABA-mediated stomatal closure in Arabidopsis. Plant Cell 21: 2357-2377
Zhao J, Wang XM (2004) Arabidopsis phospholipase D alpha 1 interacts with the heterotrimeric
G-protein alpha-subunit through a motif analogous to the DRY motif in G-protein-coupled receptors. J
Biol Chem 279: 1794-1800
Zhu JK (2016) Abiotic stress signaling and responses in plants. Cell 167: 313-324
Zou JJ, Li XD, Ratnasekera D, Wang C, Liu WX, Song LF, Zhang WZ, Wu WH (2015) Arabidopsis
CALCIUM-DEPENDENT PROTEIN KINASE8 and CATALASE3 function in abscisic acid-mediated signaling
and H2O2 homeostasis in stomatal guard cells under drought stress. Plant Cell 27: 1445-1460
Figure legends
Figure 1. The roles of reactive oxygen species (ROS) in abscisic acid (ABA)
signaling in stomatal movement
Drought stress induces the biosynthesis and/or accumulation of ABA in guard cells,
46
which is perceived by PYR1/PYL/RCAR ABA receptors. ABA-bound PYL interacts
with and inhibits clade A PP2Cs such as ABA INSENSTIVE1 (ABI1) and ABI2,
activating protein kinases such as OPEN STOMATA1 (OST1) and GUARD CELL
HYDROGEN PEROXIDE-RESISTANT1 (GHR1). After interacting with PYLs,
ABI1 is degraded by PUB12/13 E3 ligases. ABI1 dephosphorylates SLOW ANION
CHANNEL-ASSOCIATED1 (SLAC1) and inhibits its activity. OST1 phosphorylates
respiratory burst oxidase homolog protein F (RBOHF) to produce apoplastic O2∙-,
which is converted to H2O2 by superoxide dismutase (SOD). The apoplastic H2O2
signal is transduced into the cytosol by GHR1 through an unidentified mechanism to
activate a putative, unidentified calcium channel on the plasma membrane. After
binding to Ca2+, CIPK26 can phosphorylate RBOHF. Ca2+-activated CPK4/6/21/23
can phosphorylate and activate SLAC1. Apoplastic H2O2 enters the cell through the
aquaporin protein PIP2;1. CPK8 phosphorylates and enhances the activity of
CATALASE3 (CAT3). H2O2 in the cytosol may inhibit the activity of ABI1, ABI2 or
GLUTATHIONE PEROXIDASE3 (GPX3) or induce NO production via nitrate
reductases (NRs). Oxidized GPX3 inhibits ABI2 activity. Nitric oxide (NO) mediates
the S-nitrosylation of OST1 and inhibits its activity. RBOHF can also be regulated by
the G-protein α-subunit GPA1 and phosphatidic acid (PA) produced by phospholipase
Dα1 (PLDα1). KAT1, the inward-rectifying K(+) channel; GORK, guard cell outward
potassium channel; AHAs, Arabidopsis P-ATPases. Blue arrows indicate positive
regulation; red bars indicate inhibition; dotted lines indicate uncertain regulation.
Figure 2. Crosstalk among CO2, ABA and reactive oxygen species (ROS)
signaling during stomatal regulation
47
Elevated CO2 levels promote stomatal closure, which is enhanced by abscisic acid
(ABA)-mediated ROS production. CO2 in the apoplast is transported into the cytosol
by the aquaporin PIP2;1. PIP2;1 directly interacts with carbonic anhydrase βCA4 and
might channel CO2 to βCA4 to produce bicarbonate (HCO3. HCO3- signaling might
activate MPK12/4 to repress HT1. Repressed HT1 would then release its
phosphorylation and the inhibition of GUARD CELL HYDROGEN
PEROXIDE-RESISTANT1 (GHR1) or OPEN STOMATA1 (OST1), resulting in the
activation of SLOW ANION CHANNEL-ASSOCIATED1 (SLAC1) or releasing its
direct inhibition on SLAC1. HCO3- might also activate ABA signaling to enhance
SLAC1 activity by GHR1 and OST1 or Ca2+-activated CPK3/23 or directly regulate
SLAC1 via an unknown mechanism. ABA signaling induces H2O2 production
through respiratory burst oxidase homolog protein D or F (RBOHD/F). H2O2
mediates nitric oxide (NO) production through the action of nitrate reductases (NRs).
Blue arrows indicate positive regulation; red bars indicate inhibition; dotted lines
indicate uncertain regulation.
Figure 3. The roles of reactive oxygen species (ROS) in stomatal immunity
During the stomatal immunity response, the flg22 peptide is perceived by the
FLAGELLIN-SENSING2 (FLS2)– BRI1-ASSOCIATED RECEPTOR KINASE
(BAK1) co-receptor complex, which activates BOTRYTIS-INDUCED KINASE1
(BIK1). BIK1 phosphorylates the N-terminus of respiratory burst oxidase homolog
protein D, thereby activating it to produce apoplastic H2O2. BIK1 might also activate
an unknown Ca2+ channel to increase cytosolic Ca2+ levels, which would in turn
48
activate CPK28 to inhibit BIK1 or other calcium-dependent protein kinases (CPKs)
such as CPK5 to phosphorylate and activate RBOHD. Without flg22 stimulation, the
heterotrimeric G proteins composed of the non-canonical Gα proteins XLG2/3, Gβ
protein AGB1 and Gγ proteins AGG1/2 interacts with FLS2-BIK1 complex. After
perceiving flg22, the activated BIK1 phosphorylates the N-terminus of XLG2, which
leads to G proteins to dissociate from receptor complex and increase RBOHD activity
in an unknown manner. BR-SIGNALING KINASE1 (BSK1) interacts with FLS2 to
generate a flg22-induced ROS burst. In the apoplast, PRX33/34, which are regulated
by the cytokinin pathway, produce apoplastic O2∙-. Apoplastic H2O2 is transported into
the cytosol by PIP1;4, which further induces the production of nitric oxide (NO). NO
mediates the S-nitrosylation of RBOHD and ASCORBATE PEROXIDASE1 (APX1),
thereby inhibiting RBOHD activity but increasing APX1 activity. Blue arrows
indicate positive regulation; red bars indicate inhibition; dotted lines indicate
uncertain regulation.
Figure 4. Crosstalk of reactive oxygen species (ROS) between organelles
High levels of ROS are produced in chloroplasts under high-light conditions and
drought stress. FLUORESCENT IN BLUE LIGHT (FLU) and CHLORINA1 (CHL)
specifically regulate the accumulation of 1O2 in photosystem II (PSII). The
chloroplast-localized proteins EXECUTER1 (EX1) and EX2 transduce 1O2 signals to
the nucleus to regulate cell death-related genes. PSI mainly produces O2∙−/H2O2 under
stress conditions, which can be decomposed by thylakoid-bound ascorbate peroxidase
(tAPX). H2O2 has an antagonistic effect on 1O2 signaling in chloroplasts. H2O2
49
diffuses into the cytosol to activate OXIDATIVE SIGNAL-INDUCIBLE1 (OXI1),
which is required to fully activate MPK3/6. Singlet oxygen may also go through the
OXI1 and jasmonate pathways to regulate cell death. The pathogen cysteine protease
effector HopN1 directly mediates the degradation of PsbQ in PSII. The detection of
pathogen-associated molecular patterns (PAMPs) leads to an increase in transient
Ca2+ levels, which is mediated by calcium-sensing receptors (CAS) in chloroplasts,
likely via the MAPK cascade. Carotenoids quench 1O2 to become β-cyclocitral, which
induces the expression of 1O2-responsive genes. Plastidial methylerythritol
cyclodiphosphate (MEcPP) and triose phosphate from chloroplasts can also induce
selected stress-responsive nucleus-encoded plastidial proteins. SAL1
dephosphorylates 3′-phosphoadenosine 5′-phosphate (PAP) to adenosine
monophosphate (AMP), which is negatively regulated by H2O2. PAP is bound by and
inhibits nuclear 5′-to-3′ exoribonucleases (XRNs), resulting in the upregulation of
various abscisic acid (ABA)-responsive genes. Chloroplasts communicate with the
mitochondria and peroxisomes through MOSAIC DEATH1 (MOD1)-mediated fatty
acid biosynthesis. In the mitochondria, ABA signaling negatively regulates Complex I
activity through negatively regulating the ABA OVERLY SENSITIVE6 (ABO6) and
ABO8 in Complex I. In peroxisomes, glycolate oxidase (GOX) and acyl-CoA oxidase
(ACX) are the main enzymes that function in H2O2 production. Blue arrows indicate
positive regulation or production; red bars indicate inhibition; dotted lines indicate
uncertain regulation.
Figure 5. Reactive oxygen species (ROS) and Ca2+ waves function in cell-cell
50
communication
Apoplastic ROS mediated by respiratory burst oxidase homolog protein D (RBOHD)
triggers the generation of Ca2+ transients via the activity of unknown receptor-like
kinases, or they enter neighboring cells via water channels. Osmotic stress activates
OSCA1 (REDUCED HYPEROSMOLARITY, INDUCED CA2+ INCREASE1) to
increase Ca2+ levels in the cytosol. In turn, Ca2+ activates protein kinases such as
CPK5 to phosphorylate and activate RBOHD, thus forming ROS and Ca2+ waves that
function in cell-to-cell and long-distance communication. The vacuolar
cation-permeable channel TWO PORE CHANNEL1 (TPC1) and plasma
membrane-localized glutamate receptor-like proteins (GLRs) may be involved in
increasing the levels of Ca2+. Ca2+ can diffuse into neighboring cells through
plasmodesmata (PD). Blue arrows indicate positive regulation or production; dotted
lines indicate uncertain regulation.
51
Figure. 1
Figure. 2
52
Figure. 3
Figure. 4
53
Figure. 5